Free Radical Biology and Medicine 75 (2014) 69–83

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Free Radical Biology and Medicine

journal homepage: www.elsevier.com/locate/freeradbiomed

Review Article

The active role of vitamin C in mammalian iron metabolism:

Much more than just enhanced iron absorption!
Darius J.R. Lane n, Des R. Richardson n
Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, University of Sydney, Sydney, NSW 2006, Australia

art ic l e i nf o a b s t r a c t

Article history: Ascorbate is a cofactor in numerous metabolic reactions. Humans cannot synthesize ascorbate owing to
Received 13 May 2014 inactivation of the gene encoding the enzyme L-gulono-γ-lactone oxidase, which is essential for ascorbate
Received in revised form synthesis. Accumulating evidence strongly suggests that in addition to the known ability of dietary
4 July 2014
ascorbate to enhance nonheme iron absorption in the gut, ascorbate within mammalian systems can
Accepted 8 July 2014
Available online 15 July 2014
regulate cellular iron uptake and metabolism. Ascorbate modulates iron metabolism by stimulating ferritin
synthesis, inhibiting lysosomal ferritin degradation, and decreasing cellular iron efflux. Furthermore,
Keywords: ascorbate cycling across the plasma membrane is responsible for ascorbate-stimulated iron uptake from
Ascorbate low-molecular-weight iron–citrate complexes, which are prominent in the plasma of individuals with iron-
Vitamin C overload disorders. Importantly, this iron-uptake pathway is of particular relevance to astrocyte brain iron
Iron
metabolism and tissue iron loading in disorders such as hereditary hemochromatosis and β-thalassemia.
Transferrin
Recent evidence also indicates that ascorbate is a novel modulator of the classical transferrin–iron uptake
Ferritin
Dcytb
pathway, which provides almost all iron for cellular demands and erythropoiesis under physiological
IRP conditions. Ascorbate acts to stimulate transferrin-dependent iron uptake by an intracellular reductive
HIF mechanism, strongly suggesting that it may act to stimulate iron mobilization from the endosome. The
Free radicals ability of ascorbate to regulate transferrin iron uptake could help explain the metabolic defect that
contributes to ascorbate-deficiency-induced anemia.
& 2014 Elsevier Inc. All rights reserved.

Contents

Ascorbate biochemistry in mammals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

Cellular ascorbate uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Sodium-dependent vitamin C transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Facilitative glucose transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Iron uptake, storage, and efflux: new roles for ascorbate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Non-transferrin-bound iron uptake and ascorbate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
NTBI and ferrireductases: Dcytb and the cytochromes b561 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Dcytb as a ubiquitous oxidoreductase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Ascorbate efflux and ferrireduction: a novel paradigm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Transferrin iron uptake and ascorbate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Ascorbate stimulates iron uptake by an intracellular reductive mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
Ascorbate may act at the level of transferrin-cycle endosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
Cellular iron storage, efflux, and homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Ferritins and iron storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

Abbreviations: Asc, L--ascorbate; CGcytb, chromaffin granule cytochrome b561; Dcytb, duodenal cytochrome b561; DHA, dehydroascorbate; DMT1, divalent metal
transporter isoform 1; FBXL5, F-box and leucine-rich repeat protein 5; FIH, factor inhibiting HIF; FPN1, ferroportin 1; FTH1, H-ferritin; FTL, L-ferritin; GULO, L-gulono-γ-
lactone oxidase; GLUT, facilitative glucose transporter; HIF, hypoxia-inducible factor; holo-Tf, mono- or diferric Tf; IRE, iron-responsive element; IRP, iron-regulatory protein;
ISC, iron–sulfur cluster; Lcytb, lysosomal cytochrome b561; NTBI, non-Tf-bound iron; PHD, prolyl-4-hydroxylase domain-containing iron-dependent prolyl hydroxylase;
SDR2, stromal cell-derived receptor 2; Steap3, six-transmembrane epithelial antigen of the prostate-3; SVCT, sodium–vitamin C cotransporter; Tf, transferrin; TfR, Tf
receptor; UTR, untranslated region
n
Corresponding authors. Fax: þ 61 2 9351 3429.
E-mail addresses: darius.lane@sydney.edu.au (D.J.R. Lane), d.richardson@med.usyd.edu.au (D.R. Richardson).

http://dx.doi.org/10.1016/j.freeradbiomed.2014.07.007
0891-5849/& 2014 Elsevier Inc. All rights reserved.
70 D.J.R. Lane, D.R. Richardson / Free Radical Biology and Medicine 75 (2014) 69–83

Systemic iron homeostasis: cellular iron efflux, hepcidin,

and ferroportin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Iron homeostasis: the iron-regulatory protein (IRP)–iron-responsive element (IRE) system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Roles for ascorbate in regulating iron storage, efflux, and the IRP–IRE system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Iron homeostasis: the HIF system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Roles for ascorbate in regulating the HIF system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Some important caveats: cells in culture versus cells in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

Ascorbate biochemistry in mammals

It is well known that L-ascorbate (Asc)1 is a physiological
reductant and enzyme cofactor that contributes to numerous well-
defined enzymatic reactions involving collagen hydroxylation, carni-
tine and norepinephrine biosynthesis, tyrosine metabolism, and
peptide hormone amidation [1–4]. Ascorbate also contributes sig-
nificantly to cellular antioxidation as a water-soluble chain-breaking
radical scavenger [5–7] and to the recycling of plasma membrane
α-tocopherol (vitamin E) by reducing the α-tocopheroxyl radical
[3,8–10]. Ascorbate also recycles tetrahydrobiopterin [11,12], which is
important for nitric oxide synthase and tyrosine hydroxylase activ-
ities. The recycling of vitamin E by Asc is important for protection
against lipid peroxidation in membranes [8–10,13]. Most mammals
are capable of de novo hepatic synthesis of Asc from glucose, through
a biosynthetic pathway that employs the enzyme L-gulono-γ-lactone
Fig. 1. Biological redox reactions of ascorbate. The Asc monoanion is the pre-
dominant species of vitamin C present under physiological conditions. Asc under-
goes a thermodynamically favorable and reversible one-electron oxidation to the
ascorbyl radical (AR; Reaction (1)). The AR is stabilized by resonant distribution of
the resultant unpaired electron over the ring structure and can be enzymatically
recycled back to Asc. If not rapidly reduced, two ARs can dismutate to form one
molecule each of the two-electron oxidized form, dehydroascorbate (DHA; Reac-
tion (2)) and Asc. DHA can be reduced within the cell interior back to Asc in a two-
electron reduction, which bypasses the AR (Reaction (3)). DHA is very unstable and,
if not rapidly reduced back to Asc, undergoes an essentially irreversible hydrolytic
ring opening to 2,3-diketogulonic acid with a half-life of several minutes under
physiological conditions (Reaction (4)). Adapted from Lane and Lawen, [78].
oxidase (GULO) for the terminal oxidation reaction [14–17]. However,
higher primates (including humans), guinea pigs, and some bats are
obligatorily dependent on dietary sources of the vitamin [18,19]. This
requirement is due to an inactivation of the GULO gene [14,15,20].
After absorption from the intestinal lumen in humans, Asc is
transported in the blood and is typically found at millimolar
concentrations intracellularly (nucleated cells) and at micromolar
concentrations in extracellular fluids and erythrocytes [21–23].
Under physiological conditions, Asc typically undergoes a reversible
one-electron oxidation to the ascorbyl radical (also known as semi-
dehydroascorbate, monodehydroascorbate, or the ascorbate free radi-
cal; Fig. 1). The ascorbyl radical is relatively stable [6] and can be
enzymatically recycled to Asc (see below). If they are not rapidly
reduced, two ascorbyl radicals can dismutate to one Asc and one
dehydroascorbate (DHA) molecule [8,17,20] (Fig. 1). The dismutation of
two ascorbyl radicals leads to Asc being able to effectively donate two
reducing equivalents in distinct monoelectronic cellular redox reac-
tions [5]. Within cells, DHA is rapidly reduced back to Asc by
glutathione (GSH)- and NAD(P)H-dependent enzymatic and none-
nzymatic reactions [17]. The mitochondrial electron transport chain is
also an important site of intracellular DHA reduction in nucleated cells
[24–29], with reduction taking place at an apically exposed section of
complex III in mammals [26]. The reduction of DHA appears to occur
as a two-electron step that bypasses the intermediate production of
the ascorbyl radical [8]. Intracellular Asc regeneration from either the
ascorbyl radical or DHA has been reviewed by Linster and Van
Schaftingen [17] and Du et al. [30] and is not described in detail herein.
Importantly, DHA is structurally unstable, with a half-life of
several minutes under physiological conditions [31–33]. The rapid
degradation of DHA by ring opening to diketogulonic acid is
irreversible in mammalian systems [31–33] (Fig. 1). This property
has probably been an evolutionary driving force in biochemical and
cellular adaptations to Asc auxotrophy in GULO-deficient species.
Indeed, under physiological conditions, vitamin C is maintained
predominantly in its fully reduced form in both intra- and extra-
cellular biological fluids. As already alluded to, mammalian cells
possess a variety of conservative reductive mechanisms for main-
taining both intra- and extracellular Asc [8,17,20,34,35]. Even
cultured cells, which are chronically Asc-deficient owing to the lack
of supplementation and/or biosynthesis under standard culture
conditions [23,36,37], still maintain a high capacity for Asc regen-
eration. The net effect of this regeneration is the maintenance of
physiological Asc concentrations within biological fluids (e.g.,
plasma, interstitial and cerebrospinal fluid) at the expense of other,
predominantly intracellular, reductants (e.g., GSH and NAD(P)H).
Cellular ascorbate uptake
The majority of mammalian cells, with the notable exception of
human erythrocytes [21], maintain intracellular Asc concentrations
that are higher (e.g., up to 30-fold in some cases) than those in the
extracellular fluid [8,20,22]. For instance, lymphocytes accumulate
 D.J.R. Lane, D.R. Richardson / Free Radical Biology and Medicine 75 (2014) 69–83 71

intracellular Asc to concentrations of approximately 4 mM in the Sodium-dependent vitamin C transporters

context of plasma concentrations of 40–80 μM [38]. Further, neu-
rons maintain intracellular Asc concentrations of up to 10 mM,
whereas extracellular concentrations of Asc are maintained at
200–400 μM [39,40]. This outward-facing concentration gradient is
generated predominantly by sodium-dependent import of Asc into
cells by sodium–vitamin C cotransporters (SVCTs) 1 and 2
[4,22,41,42], which utilize the sodium concentration gradient across
the plasma membrane (Fig. 2A).
Human SVCT1 (KM range: 65–237 μM) is expressed mostly in
apical membranes of intestinal epithelial cells and renal tubular
cells (assisting in absorption and reabsorption of the vitamin,
respectively), whereas human SVCT2 (KM range: 8–62 μM) is
expressed in most other nucleated cells of the body [41]. The
expression of SVCT2 within the brain is vital for brain function and
Asc homeostasis in this organ [4]. The result of near-ubiquitous
SVCT2 expression throughout the body is that trans-plasma
membrane Asc transport by this transporter is close to saturation
at the Asc levels found in plasma and extracellular fluid.
The SVCTs are crucial for the maintenance of intracellular Asc
concentrations in most nucleated cell types [42,43]. SVCT1 is
mainly expressed in epithelial tissues (e.g., intestinal epithelial
cells), where it plays a major role in regulating whole-body Asc
levels [43]. In contrast, SVCT2 has a more widespread expression
and is largely responsible for cellular loading with Asc against a
concentration gradient in most tissues [43].
The regulation of SVCTs is known to occur at both the transcrip-
tional and the posttranslational level (e.g., glycosylation and phos-
phorylation) [43]. Importantly, SVCT1 and SVCT2 expression is
inversely regulated by intracellular Asc levels. Indeed, in Gulo  / 
mice that are unable to synthesize their own Asc [44], the depletion
of Asc results in increased hepatic SVCT1 and SVCT2 expression at
the mRNA and/or protein levels [45,46]. Notably, the expression of
SVCT2 is also regulated by nitric oxide in an NF-κB-dependent
manner, which probably occurs through cGMP/PKG-dependent
regulation of NF-κB transcriptional activity [47]. Additionally, SVCT2
expression can be regulated in endothelial cells by oxidized low-
density lipoprotein in a manner that is preventable by Asc [48].

Fig. 2. Routes of ascorbate import in mammalian cells. (A) Two main pathways
exist for Asc import into mammalian cells. The Asc pathway (1) imports Asc in a
sodium-dependent manner via SVCTs (i.e., SVCTs 1 and 2) in many nucleated cells.
The DHA pathway (2) imports DHA via GLUTs (i.e., GLUTs 1, 3, and 4) in a sodium-
independent manner. (B) In erythrocytes from Asc auxotrophs (e.g., humans) the
association of the integral plasma membrane protein stomatin (band 7.2b) with
GLUT1 changes the import preference from D-glucose to L-DHA uptake, resulting in
DHA transport that is not competitively inhibited by physiological glucose
concentrations. In the case of DHA import, intracellular DHA is rapidly reduced
to Asc by a variety of enzymatic and nonenzymatic reductive mechanisms that rely
on NADPH and GSH as sources of reducing equivalents. Adapted from Lane and
Lawen, [78].
Facilitative glucose transporters
Cells can also accumulate intracellular Asc against a concentra-
tion gradient via lower affinity, higher capacity transport of DHA
through the facilitative GLUTs 1, 3, and 4 [22,49–52] (Fig. 2A).
Interestingly, unlike most ascorbate-producing species, human
erythrocytes can efficiently accumulate intracellular Asc from
extracellular DHA, with minimal competition from the relatively
high plasma glucose concentrations [53,54]. This adaptation arises
from the association of GLUT1 with the integral membrane protein

Fig. 3. Ascorbate and ferrireduction in non-transferrin-bound iron uptake. (A) Classical models of non-Tf-bound iron uptake postulate the requirement for a ferrireductase
that reduces extracellular iron at the expense of intracellular reducing equivalents. Dcytb may function as a ferric (Fe3 þ ) reductase, particularly in duodenal enterocytes, by
which ferric ions are reduced monoelectronically to ferrous (Fe2 þ ) ions, at the expense of intracellular Asc. (B) Dcytb may also function as an ascorbyl radical (AR) reductase,
helping to preserve extracellular Asc. (C) A recently described, novel mechanism for extracellular ferrireduction in mammals involves release of Asc, possibly by volume-
sensitive anion channels, that has been formed intracellularly as a result of DHA import and subsequent reduction back to Asc. Nascently effluxed Asc then reacts directly
with extracellular non-Tf-bound ferric iron, reducing it to ferrous iron that can be imported by ferrous-selective transporters. The precise mechanisms and proteins involved
in ascorbate efflux remain to be identified. Adapted from Lane and Lawen, [78].
72 D.J.R. Lane, D.R. Richardson / Free Radical Biology and Medicine 75 (2014) 69–83
stomatin (band 7.2b) [55] and appears to be a compensatory
regenerative mechanism for the lack of endogenous Asc produc-
tion [55–59] (Fig. 2B). With respect to DHA uptake by cells, an
inward-facing DHA gradient is maintained by the rapid reduction
of imported DHA back to Asc, which largely occurs in an NADPH-
and GSH-dependent manner [22,53,60].
Iron uptake, storage, and efflux: new roles for ascorbate
Iron is vital for cellular survival, as evidenced by the onset of cell
death after excessive iron depletion [61,62]. Almost all organisms
require iron for metabolism and it plays a multitude of roles in
bacteria, plants, and animals. Adult humans have 3–5 g of iron in the
body [63], 480% of which is found in the hemoglobin of erythro-
cytes and 10–15% in muscle myoglobin and other iron-containing
proteins and enzymes. Typically, only 0.1% circulates in the plasma
bound to the major plasma iron-binding protein, transferrin (Tf)
[61]. Cellular iron storage predominantly occurs within protein
nanocages created by ferritin [64]. Whereas most iron in mamma-
lian systems is contained within the oxygen-binding proteins
hemoglobin and myoglobin, many other cellular proteins also rely
on iron for their function. These include other heme-containing
proteins and enzymes (hemoproteins; e.g., the cytochromes) as well
as iron–sulfur cluster (ISC)-containing proteins (e.g., succinate dehy-
drogenase) [61] and nonheme, non-ISC, iron-containing proteins
(e.g., iron- and 2-oxoglutarate-dependent dioxygenases).
Iron that is improperly sequestered, however, can be highly toxic to
cells. This occurs predominantly through its ability to act as a pro-
oxidant. Indeed, redox-active iron can catalyze the production of
reactive oxygen species through the Fenton [65] and Haber–Weiss
[66] reactions, resulting in the formation of highly damaging hydroxyl
radicals [61]. As too much or too little iron is detrimental, cellular iron
homeostasis must be tightly controlled through the regulation of
import, storage, and efflux [61,67,68]. Far from the classical view that
Asc is merely a passive dietary factor that enhances nonheme dietary
iron absorption, a growing body of evidence indicates Asc is an active
molecular participant in cellular, and perhaps systemic, iron metabo-
lism. These findings are reviewed below.
Non-transferrin-bound iron uptake and ascorbate
In its physiological form, extracellular iron is typically com-
plexed by low-molecular-weight (Mr) ligands or iron-binding
proteins. The most important iron complexants in the extracellular
fluids of mammalian systems are Tf (see below) and citrate [61,68].
Although the basic mechanisms involved in the uptake of iron are
quite well understood, the mechanisms of ferrireduction in both
Tf- and non-Tf-bound iron (NTBI) uptake by mammalian cells
remain unclear [61].
Virtually all iron in plasma is bound to Tf under physiological
conditions [61] (see below for further discussion). However, in
diseases resulting in iron overload (e.g., hereditary hemochroma-
tosis and β-thalassemia), Tf can become saturated with iron, such
that excess plasma iron occurs in the circulation as NTBI [69].
Although the precise biochemical nature of NTBI is ill defined [70],
the term typically refers to a putative low-Mr pool of iron bound to
small organic complexants, such as citrate and ATP [70,71]. This
pool of NTBI is present at very low concentrations in extracellular
biological fluids such as plasma and interstitial fluid [72,73].
However, it becomes significant in the context of iron-overload
diseases such as hereditary hemochromatosis, hypotransferrine-
mia, and hemolytic anemias (e.g., β-thalassemia), in which plasma
iron levels increase and may exceed Tf-binding capacity [72,73].
Low levels ( o1 μM) of NTBI have been documented in healthy
individuals, but may rise up to 10–20 μM under conditions of
severe iron overload [70].
NTBI and ferrireductases: Dcytb and the cytochromes b561
In most instances, iron uptake from NTBI can be blocked by
extracellular iron(II) chelators [74–76], which supports the notion
that extracellular iron(III) must first be reduced to iron(II) before
uptake. In fact, it was originally hypothesized that a plasma
membrane ferrireductase was responsible for NTBI reduction
before uptake by mammalian cells [77,78] (Fig. 3A). However,
identification of the enzyme(s) responsible for cellular ferrireduc-
tion before iron uptake has been the subject of much controversy.
Dcytb (also known as CYBRD1) is an iron-regulated ferrireduc-
tase in the striated border membrane of duodenal enterocytes,
which has been suggested to be responsible for nonheme iron
reduction during dietary iron absorption [79]. This hypothesis was
subsequently challenged by the observation that Cybrd1  /  (i.e.,
Dcytb-knockout) mice did not develop iron deficiency on a
standard lab diet, or greater iron deficiency on an iron-deficient
diet, compared to wild-type mice [80]. These results suggest that
Dcytb's activity may be supplemented by the action of other
ferrireductases or ferrireductants. As the expression of Dcytb is
iron regulated [81–84], and as the expression of this protein
clearly stimulates iron uptake in vitro [85], Dcytb is likely to play
some role (albeit probably a conditionally redundant one), in
nonheme iron absorption.
Dcytb is a member of the cytochrome b561 family, which exists in
all eukaryotic kingdoms [86]. Cytochrome b561 (also known as
chromaffin granule cytochrome b561; CGcytb/CYB561/CYB561A1)
catalyzes transmembranous electron transfer from cytosolic Asc to
intravesicular ascorbyl radicals in neuroendocrine secretory gran-
ules [87].
CGcytb was initially recognized as a redox-active component of
catecholamine storage granules in the early 1960s [88]. Flatmark and
others demonstrated that this activity was the result of a unique
heme-containing cytochrome located in the membranes of these
vesicles [89–91]. In the 1980s, spectroscopic and EPR studies demon-
strated that this cytochrome was responsible for equilibrating the
ascorbate–ascorbyl radical pair inside the granule lumen with this
redox pair in the cytosol [92–94]. Indeed, CGcytb is canonically
involved in transmembranous electron transfer from cytosolic Asc to
the intravesicular ascorbyl radical in neuroendocrine secretory gran-
ules [87], which occurs by a series of concerted electron/proton
transfers across the membrane [95,96]. The resulting Asc is subse-
quently oxidized by copper-containing dopamine β-hydroxylase and
peptidylglycine α-amidating monooxygenase [97]. The CGcytb-
catalyzed reaction involves a histidine cycling mechanism of coupled
proton/electron transfer between Asc and ascorbyl radical [98]. Addi-
tional members of the cytochrome b561 family of enzymes in
mammals include lysosomal cytochrome b561 (Lcytb/CYB561A3)
[86,99], stromal cell-derived receptor 2 (SDR2/FRRS1) [100,101], and
the putative tumor suppressor 101F6 (CYB561D2/TSP10) [102,103].
Notably, Dcytb, Lcytb, CGcytb, 101F6, and SDR2 are all capable of
stimulating cellular ferrireduction and are expressed at the plasma
membrane as well as at intracellular sites [79,86,100,104,105].
Although the bulk of evidence suggests that Asc is the major
electron donor for these proteins [106], a recent report suggests that
dihydrolipoic acid can donate reducing equivalents to cytochrome
b561 proteins [107]. Although the cellular levels of dihydrolipoic acid
are relatively low compared to Asc, this observation may help explain
the apparent Asc-independent ferrireductase activity of Dcytb that
has been reported [105]. With the exception of Dcytb, there is still a
paucity of direct evidence directly linking any of these proteins to
endogenous extracellular or intracellular ferrireduction reactions
in vitro or in vivo. Although much evidence suggests likely roles
 D.J.R. Lane, D.R. Richardson / Free Radical Biology and Medicine 75 (2014) 69–83
for some of the cytochrome b561 family members in mammalian
ferrireduction, the precise cellular functions of these proteins remain
unclear.
Dcytb as a ubiquitous oxidoreductase
In addition to being implicated as a ferrireductase at the
striated border membrane of duodenal enterocytes [79,108], Dcytb
has also been suggested to function as an oxidoreductase in a
variety of tissue and cell types including human erythrocytes
[109], lung epithelial cells [105], K562 cells [110,111], HepG2 cells
[111], Caco-2 cells [111], and astrocytes [112,113]. This observation
suggests a more general function for Dcytb than one of just
enhancing dietary nonheme iron absorption. In support of this
notion, a growing number of studies [79,82,104,105,114–116] have
implicated Dcytb as a ferrireductase in the cellular reduction of
NTBI. Importantly, as Dcytb demonstrates partial conservation of
the canonical Asc-binding motif originally identified in CGcytb
[86,97], Asc is a likely proximal electron donor for this activity,
although dihydrolipoic acid may also be involved [107]. Accord-
ingly, several studies in which Dcytb-expressing cells have been
supplemented with Asc or DHA provide support for this conclu-
sion [82,86,104,116].
Ascorbate efflux and ferrireduction: a novel paradigm
The ferrireductase-dependence hypothesis for extracellular
ferrireduction has been challenged by studies showing that NTBI
reduction can occur owing to efflux of reductants such as super-
oxide [117], dihydrolipoic acid [118], and Asc [76,119–121]. We and
others have demonstrated that extracellular Asc oxidase, which
rapidly degrades Asc to DHA, abolished the reduction of iron(III)
citrate by Asc-loaded cells, as well as greatly inhibiting Asc-
loading-dependent iron uptake [76,119,121]. This sensitivity
clearly indicates a requirement for effluxed Asc in the reduction
of NTBI before uptake. Importantly, our data indicate that Asc is
exported from the cells and directly reduces low-Mr iron(III)
citrate complexes to iron(II) for cellular uptake, with the latter
occurring via an iron(II)-selective transporter such as divalent
metal transporter isoform 1 (DMT1) [119,122–125].
Regarding the mechanisms of ascorbate efflux from mammalian
cells (for reviews see [22,77,78,126]), several plausible candidates for
a release pathway have been proposed [22]. These include
(i) exocytosis of ascorbate [22,127], (ii) connexin hemichannels
[128], and (iii) plasma membrane (volume-sensitive) anion channels
(VSOACs) [22,129–131]. The VSOAC hypothesis for ascorbate efflux is
perhaps the best supported by experimental data [22]. These
functionally defined channels are thought to be plasma membrane
anion channels that are involved in regulatory volume decrease of
cells after cellular swelling caused by hypotonic shock [132] or
glutamate-transporter-dependent [133] glutamate/aspartate uptake
by astrocytes [130,134]. Electrophysiological evidence demonstrates
that VSOACs are permeable to ascorbate [132], although the mole-
cular identities of many of these channels remain uncertain. More-
over, the observation that both VSOAC permeability [135] and
ascorbate efflux from cells [22,76,129] can often be blocked by
nonspecific, sulfonate anion channel inhibitors, such as 4,40 -dii-
sothiocyanatostilbene-2,20 -disulfonic acid and 4-acetamido-40 -iso-
thiocyanostilbene-2,20 -disulfonic acid, suggests that a significant
proportion of ascorbate release occurs via these channels. Ascorbate
efflux, which is highly sensitive to sulfonate anion channel inhibitors,
occurs in a variety of cell types, including astrocytes [130,133,136],
hepatocyte-like HepG2 cells [137], SH-SY5Y neuroblastoma cells
[131], coronary artery endothelial cells [138], and K562 cells [76],
but not in endothelial cells [139]. Importantly, not all anion channels
are sensitive to sulfonate inhibitors [140]. In the case of endothelial
73
cells, the release of ascorbate is largely insensitive to such inhibitors
[138,139], but appears to be stimulated by Ca2 þ and ATP and
inhibited by gluconate [138,141]. Taken together, these results
suggest that ascorbate efflux in endothelial cells involves Ca2 þ -
dependent anion channels that are regulated by the G-protein-
coupled purinergic P2Y2 receptors [138,141]. However, as previously
suggested, these pharmacological properties may also suggest a
Ca2 þ -dependent, P2Y2-regulated exocytotic mechanism [78]. In the
case of VSOAC involvement in ascorbate efflux from astrocytes and
other cell types, a plasma membrane isoform of the voltage-
dependent anion channel 1 [142,143] is a plausible candidate that
should be seriously considered. Clearly, further molecular studies are
needed to resolve the involvement of these pathways.
We have proposed a model of ascorbate-dependent NTBI
reduction and uptake by mammalian cells in which iron uptake
is preceded by iron reduction by extracellular Asc, which is
subsequently regenerated from DHA by trans-plasma membrane
Asc cycling [76–78,119] (Fig. 3C). Notably, a recent study has
identified an analogous Asc efflux mechanism as a novel strategy
for iron reduction and transport in plants [144]. Indeed, Pisum
sativum and Arabidopsis embryos exhibit ferrireduction activity
that is not catalyzed by the typically attributed membrane ferrir-
eductase activity, but is instead due to the efflux of high levels of
Asc that directly reduce iron(III) present in extracellular iron–
citrate–malate complexes [144].
Transferrin iron uptake and ascorbate
Under normal physiological conditions, virtually all plasma iron is
tightly bound to Tf [61]. Iron that is taken up by enterocytes is
released into the bloodstream and oxidized to the ferric state
potentially by the transmembrane multicopper ferroxidase, hephaes-
tin, which occurs predominantly in the basolateral membrane of
enterocytes [145]. The homologous soluble multicopper ferroxidase,
ceruloplasmin, which is abundant in plasma, is likely also to play
some role in this activity [146]. The iron(III) that is formed by the
action of these ferroxidases is specifically bound to serum Tf, an 80-
kDa glycoprotein that is mainly synthesized by the liver [147]. Each Tf
molecule can bind one or two iron(III) ions [148,149] to form one
molecule of mono- or diferric Tf (holo-Tf), respectively.
The binding of iron(III) to Tf occurs with high affinity in a pH-
dependent manner, with maximal binding occurring at pH 7.4
(affinity constant at atmospheric pCO2 for each binding site of
 1020 M  1) [150]. Holo-Tf binds to the integral membrane protein
Tf receptor 1 (TfR1) to donate iron to cells [61,68,147] (Fig. 4A).
TfR1 is a glycoprotein of about 90-95 kDa, which forms a homo-
dimer of approximately 180-190 kDa that is linked by disulfide
bonds [151] and is capable of binding two molecules of holo-Tf
[152]. TfR1 is expressed by most cells, with the exception of
mature erythrocytes [153] and possibly oligodendrocytes, micro-
glia, and astrocytes in vivo [154].
The affinity of TfR1 for diferric Tf at pH 7.4 is about 2000-fold
higher than for apo-Tf [149,155] and about 20-fold higher than for
either of the monoferric Tf forms [156]. The entire holo-Tf–TfR1
complex is then endocytosed via receptor-mediated endocytosis in
clathrin-coated pits [157]. The pH of the endosome decreases after
internalization, owing to the activity of an ATP-dependent proton
pump, the vacuolar-type H þ -ATPase, in the endosomal membrane
[158,159]. Owing to the resulting acidic environment (i.e., a lumen
pH of 5.3–5.6 [160]), the iron(III) dissociates from Tf, whereas apo-
Tf remains tightly bound to TfR1 [158,161].
Before the transport of iron from the endosome to the cytoplasm
by DMT1 [162,163], iron(III) that is released from Tf must first be
reduced to iron(II) [164,165]. The only ferrireductase identified to be
involved in this process is the six-transmembrane epithelial antigen
74 D.J.R. Lane, D.R. Richardson / Free Radical Biology and Medicine 75 (2014) 69–83

Fig. 4. Model of ascorbate-dependent stimulation of transferrin–iron uptake. (A) In Asc-depleted cells, holo-Tf binds to cell surface Tf receptors (TfR) and is internalized by
receptor-mediated endocytosis. The endosomes that form become acidified via the proton-pumping vacuolar-type H þ -ATPase (V-ATPase). Acidification leads to release of
iron from Tf, whereas iron-free Tf (apo-Tf) remains bound to the TfR. The intraendosomal Fe3 þ is then reduced to Fe2 þ and transported across the endosomal membrane by
the divalent metal transporter 1 (DMT1) and/or Zip14. The apo-Tf–TfR complex returns to the cell surface, where apo-Tf dissociates at neutral pH. After transport across the
endosomal membrane, iron initially enters the labile iron pool (LIP) and can be stored in ferritin. (B) In contrast, intracellular ascorbate enhances: (i) Tf-dependent iron
uptake, (ii) LIP size, (iii) ferritin synthesis, and (iv) iron deposition in ferritin. The last two effects on ferritin may be secondary to the increase in the LIP and Tf-dependent Fe
uptake. Asc appears to act intracellularly via a reductive mechanism, either by direct ferrireduction or via a transmembrane reductase. This process probably (as indicated by
dotted lines) involves direct import of Asc into the Tf cycle endosome and direct or chemical ferrireduction and/or provision of electrons to an endosomal ferrireductase to
enhance Tf-dependent delivery of iron to cells. Adapted from Lane et al. [120].

of the prostate-3 (Steap3) [166,167], but this activity may be
restricted to erythroid precursors. It should also be noted that other
iron(II) transporters have been proposed to contribute to iron
mobilization from Tf-cycle endosomes, including ZIP14 [168],
although their relative contributions in different cell types remain
to be established. Finally, the complex of apo-Tf and TfR1 is then
recycled back to the plasma membrane and the apo-Tf dissociates
from TfR1 at the slightly alkaline pH of the extracellular space [169]
(Fig. 4A).
Ascorbate stimulates iron uptake by an intracellular reductive
mechanism
We recently demonstrated that Asc regulates iron uptake from
Tf in a variety of human cell types [120]. This observation is
intriguing, as Asc is a ubiquitous and normally abundant cellular
reductant in vivo, yet it is absent under standard cell culture
conditions [170]. Significantly, typical physiological plasma Asc
concentrations enhance iron uptake by Asc-derived cells by up to
100% from Tf, which is accompanied by a corresponding increase
in cellular ferritin expression and ferritin-iron loading [120].
Although Asc can increase ferritin either by promoting de novo
synthesis [171] or by inhibiting ferritin autophagy [172] (see below
for further discussion), we demonstrated that none of these
processes were responsible for Asc's ability to stimulate
Tf-dependent iron uptake [120]. Indeed, these results support
the general notion that ferritin does not contribute to Tf-
dependent iron uptake per se, although it serves as the major
iron storage depot for nascently internalized iron.
Ascorbate may act at the level of transferrin-cycle endosomes
To gain insight into Asc's mechanism of action in stimulating Tf-
dependent iron uptake, we initially adopted a comparative approach
to examine key differences between the Asc-stimulated uptake from
iron–citrate complexes and holo-Tf [120]. We determined that, unlike
iron–citrate [76,119,121], the uptake of iron from Tf was largely
independent of: (i) the reductive action of extracellular Asc and (ii)
the extracellular labilization of iron from Tf [120]. In fact, the results of
our experiments with membrane-impermeative and membrane-
permeative Asc-oxidizing reagents strongly suggest that Asc acts
intracellularly to enhance Tf-dependent iron uptake [120].
Consistent with these findings, and with the fact that most
known biological activities of Asc are mediated by the molecule's
reducing activity [5,17], our results demonstrated that the redu-
cing ene-diol moiety of Asc was required for stimulation of iron
uptake [120]. Importantly, we also showed that this was not
due to a general increase in cellular reducing capacity in the
presence of Asc or to a specific increase in NADPH. In fact,
incubation of SK-Mel-28 cells with a physiological level of Asc
(50 μM) resulted in depletion of cellular NADPH, but not total
NADP [120]. This finding is consistent with the involvement of
NADPH and thioredoxin reductase [35,173] during intracellular
Asc recycling reactions. Taken together, our results suggest that
Asc enhances Tf-dependent iron uptake in a manner dependent
specifically on Asc's reducing activity and an intracellular pool of
the vitamin.
Of importance to the mechanism by which Asc acts to enhance
Tf-dependent iron uptake, Asc is at least as effective as NADH
in vitro at mobilizing iron from isolated Tf-containing endosomes
[164,165]. We also demonstrated that, although Asc did not affect
the kinetics of Tf cycling, the expression of human TfR1 or TfR2
was necessary for the stimulatory effect of Asc on iron uptake
[120]. As an aside, our observation that Asc stimulated iron uptake
in a TfR2-dependent manner lends support to the hypothesis that,
in addition to regulating hepcidin production and systemic iron
homeostasis [174], TfR2 contributes to iron acquisition from Tf
[175,176]. However, any direct role for TfR2 in Tf-dependent iron
uptake is likely to be minor in comparison to that of TfR1
[177,178].
Finally, using an array of well-characterized inhibitors of endocy-
tosis or endosome acidification, we demonstrated that endocytosis
 D.J.R. Lane, D.R. Richardson / Free Radical Biology and Medicine 75 (2014) 69–83
and intracellular vesicle acidification were critical for Asc-enhanced
Tf-dependent iron uptake [120]. Specifically, endocytosis was neces-
sary for stimulation to occur, whereas blockade of intracellular
vesicle acidification inhibited both control and Asc-stimulated iron
uptake similarly, although the relative stimulation provided by Asc
was not affected. Together, our recent findings demonstrate that:
(i) endocytosis is essential for Asc to stimulate Tf-dependent iron
uptake and (ii) intracellular vesicle acidification facilitates both
control and Asc-stimulated iron uptake from holo-Tf equally [120].
These observations show that Asc enhances Tf-dependent iron
uptake downstream of an endocytosis event, which is further
enhanced by intracellular vesicle acidification. Thus, Asc acts intra-
cellularly via a reductive mechanism to enhance Tf-dependent iron
delivery of iron to cells, and this appears to occur subsequent to the
TfR-dependent endocytosis of holo-Tf (Fig. 4B).
As mentioned above, our results are consistent with previous
observations that Asc efficiently promotes iron mobilization from
isolated and acidified Tf-containing endosomes [164,165]. Consid-
ering these data collectively, we have proposed a model for Asc's
activity in enhancing intraendosomal ferrireduction and iron
mobilization from the Tf-containing endosome (Fig. 4B). As sug-
gested by studies on isolated endosomes containing Tf, this could
occur by Asc uptake into the endosomal vesicle followed by
chemical ferrireduction [179] and/or by supply of reducing equiva-
lents from Asc to an endosomal ferrireductase [164,165].
Importantly, the only confirmed ferrireductase of the Tf cycle is
Steap3, which appears to be most active in this role in erythroid
cells [166]. It is unlikely that Asc contributes electrons to Steap3,
which instead appears to utilize NAD(P)H [166,167], for the
following reasons. First, we observed that NADPH levels were
decreased and not increased by incubation with Asc, probably due
to NADPH-dependent intracellular ascorbate recycling reactions
[120]. Second, we also observed that Steap3 expression was
unaffected by the vitamin [120]. Although Asc may act to stimulate
Steap3 activity indirectly, the possibility that other yet-to-be-
identified endosomal Asc-dependent ferrireductases contribute
to intraendosomal ferrireduction must now be considered.
The finding that Asc stimulates Tf-dependent iron uptake [120] is
significant for the following reasons: (i) the majority of in vitro
studies on Tf-dependent iron uptake have been with performed with
Asc-depleted cells and (ii) severe Asc deficiency in humans
(i.e., scurvy), as well as experimentally induced Asc deficiency in
guinea pigs, causes an “anemia of scurvy” [180,181]. The observation
that Asc enhances iron uptake from Tf in all human cells examined
suggests that the many previous studies on cellular iron metabolism
and Tf-dependent iron uptake may have overlooked the involvement
of this important cellular reductant. This is probably due to the fact
that most cell culture studies are performed in the absence of Asc.
Moreover, the ability of Asc to enhance iron uptake from Tf [120],
which is the major donor of iron to the erythropoietic compartment
[62], could assist in explaining the metabolic defect that contributes
to Asc-deficiency-induced anemia.
Cellular iron storage, efflux, and homeostasis
Ferritins and iron storage
In nonerythroid cells, iron that has been nascently internalized
from holo-Tf or NTBI initially enters the poorly characterized labile
iron pool, after which the majority (70–80%) is incorporated into
ferritin [182,183]. Ferritin is a typically cytoplasmic heteromeric
protein complex composed of 24 subunits that form a hollow
sphere that can store up to 4500 atoms of iron as a mineralized
ferric, phosphate, and hydroxide core [183,184]. In mammals, there
are two ferritin subunits, H-ferritin (heavy; also known as FTH1)
75
and L-ferritin (light; also known as FTL), which heteropolymerize to
form a wide range of iso-ferritins (i.e., comprising different ratios of
FTH1 to FTL) with tissue-specific distributions [184]. The iron(II)
entering ferritin is readily oxidized by the intrinsic ferroxidase
activity of H-ferritin in an oxygen-dependent manner to iron(III),
followed by nucleation and mineralization of the iron center by
L-ferritin, which is devoid of ferroxidase activity [183,184]. This
form of storage is vital for cells as it avoids adventitious Fenton- and
Haber–Weiss-type redox reactions from occurring.
To be an effective store of iron, intracellular ferritins must also be
able to release their iron. The major mechanism of iron release from
ferritin under in vivo conditions is autolysosomal proteolysis, although
proteasomal degradation of the protein can also occur [185–188].
Lysosomal degradation of ferritin requires the action of the autophagic
apparatus [189]. Interestingly, in vitro studies with isolated ferritins
indicate that reductive mobilization reactions, which can be mediated
by Asc [190,191], are involved in iron release from ferritin. The in vivo
relevance of this protein-degradation-independent release of iron
from ferritin, which occurs through the eight hydrophilic channels in
the ferritin protein [183,192,193], is unclear.
Systemic iron homeostasis: cellular iron efflux, hepcidin,
and ferroportin
Systemic iron homeostasis is controlled primarily at the level of
iron efflux by the only known iron exporter, ferroportin 1 (FPN1/
SLC40A1), from key cell types into the circulation: (i) dietary iron
absorption by duodenal enterocytes (i.e., via reduced iron efflux
into the portal circulation from duodenal enterocytes), (ii) iron
recycling by splenic macrophages (i.e., via reduced iron efflux into
the plasma after the phagocytic turnover of effete and/or damaged
erythrocytes), and (iii) iron release from iron stored within
hepatocytes (Fig. 5).
Iron homeostasis: the iron-regulatory protein (IRP)–iron-responsive
element (IRE) system
Cellular iron homeostasis is predominantly regulated at the
level of the translation of key iron metabolism proteins involved in
iron uptake, storage, and release [194,195]. The IRP–IRE system is
responsible for this regulation [196] and allows for rapid altera-
tions in the synthesis of key iron metabolism proteins in response
to intracellular iron levels [36,171]. This system depends on the
mRNA-binding proteins IRPs 1 and 2 [197,198], which posttran-
scriptionally control the expression of proteins whose mRNA
possesses an IRE [67,68] (Fig. 6). IRPs bind to IREs in the 50 - or
30 -untranslated regions (UTRs) of key mRNAs involved in iron
metabolism with high affinity in iron-depleted cells, either sup-
pressing the translation of the mRNA (i.e., mRNAs in which the IRE
is located in the 50 -UTR, e.g., FTH1 or FTL) or enhancing mRNA
stability against nuclease attack (i.e., mRNAs in which the IRE is
located in the 30 -UTR, e.g., TfR1 and DMT1-I) [61,62].
The two IRPs are homologous to each other and possess a high
degree of amino acid sequence identity (i.e., 64% in humans
[194,196]). IRPs 1 and 2 are members of the aconitase gene family
and are probably derived from gene duplication events. Despite
their homology, the molecular responses of IRPs 1 and 2 to iron
levels are significantly different. IRP1 is an intriguing bifunctional
protein that responds to intracellular iron primarily through an ISC
“IRP1/aconitase switch” mechanism [199]. Under conditions of
increased cellular iron, which can be potentiated by Asc [171], IRP1
loses its IRE-binding activity by acquiring a cubane ISC (4Fe–4S
cluster) [195]. The acquisition of this 4Fe–4S cluster converts IRP1 into
a cytosolic aconitase: an enzyme capable of catalyzing the stereo-
specific isomerization of citrate to isocitrate via cis-aconitate [200].
76 D.J.R. Lane, D.R. Richardson / Free Radical Biology and Medicine 75 (2014) 69–83

Fig. 5. Systemic iron homeostasis and the major cell types involved. Systemic iron homeostasis is primarily regulated by the hepcidin–ferroportin (FPN1) axis. The major cell
types known to be involved in regulating/consuming iron at the systemic level are shown. Duodenal enterocytes import dietary iron from several sources including nonheme
iron, which typically must be reduced at the level of the apical membrane by chemical reductants such as Asc or by plasma membrane oxidoreductases (e.g., Dcytb). It is then
subsequently imported by ferrous iron transporters such as divalent metal transporter 1 (DMT1). Both DMT1 and Dcytb can be regulated by a recently described IRP1–HIF2α
axis of control. Nascently imported iron then enters a common intracellular pool of iron within the enterocyte. (A) Under conditions of low iron, this iron can be readily
released into the circulation by the iron efflux protein, FPN1, that is localized to the basolateral membrane. This release is coupled to reoxidation of iron by the membrane-
bound ferroxidase, hephaestin (HEPH), and iron loading of Tf. Additionally, the consumption of senescent/damaged erythrocytes by specialized macrophages (e.g., splenic
macrophages) leads to the release of iron within the macrophage followed by cellular efflux by FPN1 coupled to a plasma-membrane-bound variant of the ferroxidase
ceruloplasmin (CP). Oxidized iron is then bound by circulating Tf to form holo-Tf. The Tf-bound iron is the major source of iron for virtually all cells in the body and is
primarily consumed by erythroid progenitors during erythropoiesis. (B) Under conditions of iron overload, hepatocytes “sense” the level of Tf saturation and levels of iron
stores and consequently upregulate hepcidin expression. Hepcidin is then released into the plasma where it can then bind to FPN1 (e.g., at the level of duodenal enterocytes,
splenic macrophages, and hepatocytes), thereby triggering its internalization and degradation. Adapted from Lawen and Lane, [61].

Whereas acquisition of an ISC seems to be the predominant form
of iron-mediated regulation of IRP1, iron-regulated proteasomal
degradation is the major regulatory mechanism for IRP2 [194].
IRP2, which is unable to acquire an ISC in response to increased
cellular iron, is regulated at the level of protein abundance by the
ubiquitin–proteasome system [194]. The major mechanism by which
this occurs is the IRP2-targeting E3 ubiquitin ligase complex contain-
ing F-box and leucine-rich repeat protein 5 (FBXL5), which itself is
regulated at the level of protein stability by iron and oxygen [201–
204]. Rather than being a substrate for iron- and oxygen-dependent
hydroxylases, as with other iron- and oxygen-regulated proteins (see
below), FBXL5 contains a hemerythrin-like domain that directly
incorporates iron in a di-iron center coordinated by histidine and
glutamate residues, leading to stabilization (i.e., decreased degrada-
tion) of the protein [201–204]. In turn, this leads to increased
assembly of the FBXL5 ubiquitin E3 ligase complex and increased
rates of IRP2 degradation [201–204].
Roles for ascorbate in regulating iron storage, efflux, and the IRP–IRE
system
Ascorbate has been shown to regulate ferritin expression by
multiple mechanisms [36,37,83,120,171,172,205–207]. Incubation
of cells with physiological levels of Asc has been shown to inhibit
autophagic degradation of ferritin, leading to increased steady-
state levels of the protein [172]. Unfortunately, the mechanism of
this inhibition of autophagy remains unknown, but may relate to
Asc-dependent regulation of the multiple intersecting intracellular
signaling cascades that regulate this process.
Ascorbate has also been described to stimulate ferritin synthesis,
which appears to be related to an increase in the IRP1/aconitase
“switch” mechanism [36] (Fig. 6). Indeed, Asc increases the propor-
tion of ISC-containing IRP1 that functions as a cytosolic aconitase
and, consequently, decreases the pool of the mRNA-binding form of
IRP1 [171]. It is important to note that although the combination of
Asc and holo-Tf increases both FTH1 and FTL levels, as well as
decreasing TfR1 levels, this effect does not appear to be responsible
for the ability of Asc to increase iron uptake from holo-Tf [120]. This
is based on the following observations: (i) blockade of translation
does not consistently affect Asc-stimulated iron uptake from holo-
Tf, and the effect of Asc on ferritin appears to require a source of
iron, suggesting that an increase in ferritin synthesis is downstream
of increased iron uptake; (ii) Asc causes iron-dependent TfR1
downregulation, not upregulation, which occurs downstream of
increased iron uptake; and (iii) blockade of cytosolic aconitase
activity with the aconitase inhibitor oxalomalate does not affect
Asc-stimulated iron uptake from holo-Tf [120].
In addition to decreasing the IRE-binding activity of IRP1, Asc can
also promote the proteasomal degradation of IRP2 [208,209]. The
activity of prolyl hydroxylases, such as those involved in the
hydroxylation of HIFα proteins (see below), has been implicated in
the proteasomal degradation of IRP2 [208,209]. In contrast, a recent
report indicates that physiological levels of Asc cause an increase in
IRP2 in Caco-2 cells [206]. The question of whether Asc affects the
regulation of IRP2 abundance by modulating the FBXL5-dependent
mechanism [201,202] or by an apparently distinct mechanism
involving the activity of ascorbate-stimulated iron-dependent prolyl
hydroxylases [208], remains to be determined (see below). Interest-
ingly, the regulation of IRP2 degradation by ascorbate could be
related to the well-described antioxidant activity of ascorbate
[6,7,30]. It is known that IRP2 can be stabilized in the presence of
iron by pro-oxidants such as H2O2, and this can be antagonized by
antioxidants such as ascorbate, α-tocopherol, and N-acetylcysteine
[208,209]. These findings do not discriminate between the ascorbate-
mediated redox control of the di-iron center in FBXL5 [201,202], the
apparent prolyl-hydroxylase-dependent mechanism [208], or an
alternative cysteine-dependent S-nitrosylation-regulated pathway
for IRP2 degradation [210].
In summary, by modulating IRP1 and/or IRP2, cellular Asc
status can alter the expression of key IRP–IRE-regulated proteins
and is consequently a likely modulator of cellular iron home-
ostasis. Further studies to explore this hypothesis and its ramifica-
tions for mammalian iron metabolism are essential.
Ascorbate may also be able to modulate FPN1 [206] and/or
cellular iron efflux activity [206,211]. We have shown that Asc can
decrease iron release from various cell types [120,211], although
 D.J.R. Lane, D.R. Richardson / Free Radical Biology and Medicine 75 (2014) 69–83
Fig. 6. Ascorbate and the iron-regulatory protein (IRP)–iron-responsive element
(IRE) system: posttranscriptional control of iron homeostasis. The IRP–IRE system is a
major regulator of iron metabolism at the cellular level. It is also the most rapid
responder to perturbations in intracellular iron levels. This system provides a means
of regulating the expression of proteins involved in iron storage (H-ferritin (FTH1)
and L-ferritin (FTL)), iron export (FPN1), iron uptake (TfR1, DMT1, MRCKα), the
mitochondrial citric acid cycle (ACO2), mitochondrial hemoglobinization (eALAS),
oxygen sensing (HIF2α), and cell cycle control (CDC14A). Under conditions of low
iron, the IRPs are in their IRE-binding forms. Under conditions of high iron, the IRE-
binding activity of the IRPs is decreased. IRP1 acquires an iron–sulfur cluster (ISC),
which converts the protein into a cytosolic aconitase that is incapable of binding
IREs, whereas IRP2 is targeted for degradation by the proteasome. Ascorbate can
potentiate the loss of IRE-binding activity by promoting IRP1/cytosolic aconitase
switching and IRP2 degradation. Under conditions of high iron, the binding of IRP1 or
IRP2 to cis-regulatory motifs known as IREs in the 50 -UTR of select mRNAs (i.e., those
encoding FTH1, FTL, eALAS, FPN1, HIF2α, and ACO2) serves to inhibit translation (↓)
of the mRNA by preventing ribosomal docking (translational control), whereas the
binding of IRPs to IREs in the 30 -UTRs of select mRNAs (i.e., those encoding TfR1,
DMT1, MRCKα, and CDC14A) protects the transcripts against nuclease-mediated
degradation (mRNA stability control), leading to increased protein expression (↑). In
the absence of IRPs binding to IREs, the translation of mRNAs possessing 50 IREs can
proceed (↑),whereas mRNAs possessing 30 IREs are degraded, leading to decreased
protein expression (↓). In both cases, the IRE–IRP system effectively modulates the
rate at which IRE-containing mRNAs are translated into their corresponding proteins.
Adapted from Lawen and Lane, [61].
whether this iron efflux is FPN1-dependent is unclear. Considering
this, it is notable that physiological Asc levels caused an increase in
FPN1 expression in intestinal epithelial-like human Caco-2 cells,
which was associated with an increase in IRP2 and HIF2α [206].
Although the mechanism was not identified in that study, the
increase in FPN1 may be due to the Asc-dependent IRP1/aconitase
switch mechanism, despite the observed increase in IRP2 levels.
Further studies on the regulation of FPN1 and iron efflux activity
by Asc are clearly needed.
Iron homeostasis: the HIF system
Cellular iron homeostasis is also controlled at the level of the
transcription of iron metabolism genes. A major regulator of these
changes in transcription is the HIF system, which includes the oxygen-
and iron-regulated proteins HIF1α and HIF2α [212,213] (Fig. 7).
Low oxygen tensions (i.e., hypoxia), as well as low intracellular iron
77
Fig. 7. Ascorbate and the HIF system: transcriptional control of iron homeostasis.
Cellular iron metabolism is also regulated at the transcriptional level. A major
transcriptional control mechanism involved in the regulation of certain genes
involved in iron metabolism is the hypoxia-inducible factor (HIF) system. This
system is the primary homeostatic responder to changes in oxygen (O2) tension
and is also sensitive to changes in intracellular iron and Asc levels. Under
conditions of high iron, O2, and Asc concentrations, HIF1α and HIF2α are hydro-
xylated at specific proline residues by a class of prolyl hydroxylase domain proteins
(PHDs 1–3) whose activities vary directly with the intracellular concentrations of
O2, iron, and Asc. Prolyl hydroxylation targets HIF1/2α proteins for ubiquitination
by the E3 ubiquitin ligase, von Hippel-Lindau protein (VHL), which subsequently
marks them for proteasomal degradation. Under conditions of low iron, O2, and Asc
concentrations, HIF1/2α proteins are stabilized and form heterodimers with the
constitutively expressed HIFβ protein. These heterodimers then translocate to the
nucleus and activate the transcription of specific genes that contain hypoxia-
response elements (HREs; e.g., genes encoding Tf (TF), TfR1 (TFRC), DMT1
(SLC11A2), FPN1 (SLC40A1), Cp (CP), Dcytb (CYBRD1), HO1 (HMOX1), and EPO
(EPO)). Additionally, high iron, O2, and Asc levels increase hydroxylation activity of
the asparaginyl hydroxylase known as factor inhibiting HIF1 (FIH-1). Notably, FIH
inhibits the transcriptional activity of the HIF complex by inhibiting the interaction
of the C-terminal transactivation domain of HIF1α with the transcriptional
coactivator CBP/p300. Adapted from Lawen and Lane, [61].
concentrations, activate HIF1- and HIF2-regulated transcription by the
increased formation of heterodimers of HIF1α or HIF2α and the
constitutively expressed HIF1β subunit (also known as the aryl
hydrocarbon receptor nuclear translocator) [212]. HIF1α is ubiqui-
tously expressed, whereas HIF2α has a more restricted tissue distribu-
tion [214]. The HIFα/β heterodimers form transcription factors that
regulate a wide range of genes encoding proteins that are important
for cellular oxygen homeostasis and the response to hypoxia [61,213].
Both HIF1α and HIF2α are posttranslationally regulated at
the level of protein degradation in an oxygen-dependent and
iron-dependent manner [212]. This occurs by a specific class of
2-oxoglutarate-dependent dioxygenases: the prolyl-4-hydroxylase
domain-containing iron-dependent prolyl hydroxylases (PHDs) 1–3
and the asparaginyl hydroxylase, “factor inhibiting HIF” (FIH) [213].
The PHD-type hydroxylases are fully active under conditions of 21%
oxygen, found under standard cell culture conditions (see below),
and iron repletion and they hydroxylate HIFα proteins at specific
proline residues [215,216]. Importantly, it is the strict dependence of
these hydroxylases on iron that is presumed to be largely respon-
sible for the ability of cellular iron levels to modulate HIF-regulated
gene expression [213,217,218]. Hence, low levels of cellular iron will
lead to less iron for PHD metallation, reducing PHD activity and
78 D.J.R. Lane, D.R. Richardson / Free Radical Biology and Medicine 75 (2014) 69–83
limiting the hydroxylation of HIF1α, which subsequently increases
HIF1α protein levels and its transcriptional activity. Indeed, such
iron limitation would be expected to limit the activity of other 2-
oxoglutarate oxidases in an analogous manner. The hydroxylated α
subunits of HIF are then targeted for ubiquitination by the E3
ubiquitin ligase von Hippel-Lindau tumor suppressor protein, which
directs the proteins to be degraded by the proteasome [212,217].
Roles for ascorbate in regulating the HIF system
In addition to its effects on the IRP–IRE system, Asc probably also
regulates the expression of iron metabolism proteins by affecting HIF
signaling [219,220]. In fact, Asc is known to inhibit HIF1- and 2-
dependent gene expression [220] by downregulating the 1 or 2α
subunit of HIF [219–222]. This seems to occur because Asc is required
as a cofactor for the full activity of the PHDs that hydroxylate [215]
and target HIF1α for proteasomal degradation [220,223] (Fig. 7).
In addition, it has recently been shown that in addition to
promoting HIF1α degradation, Asc also prevented HIF1α-dependent
transcriptional activity, even under conditions that allowed for HIF1α
stabilization [221]. Intriguingly, both effects appeared to be mediated
by Asc's effects on the 2-oxoglutarate- and iron-dependent dioxy-
genases, including PHDs 1–3 and the asparaginyl hydroxylase FIH.
Importantly, ascorbate acts as a cofactor for these hydroxylases by
maintaining the catalytic iron center in its functional ferrous state
[215]. As such, Asc promotes HIF1α prolyl hydroxylation, leading to
degradation by the proteasome.
Ascorbate also promotes asparginyl hydroxylation, resulting in
decreased binding of the transcriptional coactivator p300 and
inactivation of HIF1α-dependent transcriptional activity, respec-
tively [221]. These findings strongly suggest that cellular Asc
deficiency is a likely enhancer of the HIF-dependent transcrip-
tional response that is known to be activated under conditions of
iron deficiency, hypoxia, metabolic disturbance, and oxidative
stress [224].
Interestingly, although Asc is required in vitro for maximal
hydroxylation activity of the PHDs, a recent study using Asc-
depleted Gulo  / mice showed normal HIF-dependent gene expres-
sion and normal hypoxic HIF1α induction [225]. Although these
findings suggest that Asc may be dispensable in vivo for HIFα-
dependent oxygen (and perhaps iron) sensing, they should be
interpreted with caution. This is because Asc-depleted Gulo  / mice
are likely to retain intracellular Asc levels [44,220], which may be
sufficient to stimulate hydroxylase activity. The role of Asc in
regulating the HIF system and its involvement in intracellular iron
sensing and iron homeostasis require further investigation.
Some important caveats: cells in culture versus cells in vivo
It is important to note that although the vast majority of cell
culture studies are performed in incubators with an oxygen
tension equivalent to that of atmospheric oxygen levels (21%), this
is much greater than the oxygen levels experienced by most cells
in most adult tissues in vivo (e.g.,  3–5% O2) [226]. Under the
relatively “hyperoxic” conditions that cultured cells typically
experience, there are marked differences in iron metabolism that
are relevant to the role of ascorbate in regulating these processes.
First, the IRPs, which posttranscriptionally regulate expression of
various proteins, including TfR1 and ferritin, are differentially
regulated by tissue oxygen levels in vivo [227]. IRP2  /  mice
demonstrate global dysregulation of iron metabolism and develop
neurodegeneration, whereas IRP1  /  mice are largely spared [228].
This has led to the conclusion that IRP2 predominates over IRP1 in
the posttranscriptional regulation of iron metabolism. However,
whereas IRP2  /  cells demonstrate severely dysregulated iron
metabolism at mildly hypoxic in vivo oxygen concentrations, no
apparent dysregulation is observed at 21% oxygen [228]. Intrigu-
ingly, under the latter condition, the latent RNA-binding activity of
the relatively large IRP1 pool [229] in cells is activated, allowing it to
substitute for the loss of IRP2 [228]. Thus, although IRP2 is the
dominant IRP involved in regulation of cellular iron homeostasis
under in vivo oxygen concentrations, in contrast, IRP1 becomes
more important under hyperoxic conditions (i.e., 21% oxygen) and is
better able to respond to changes in cellular iron status [228].
This oxygen-dependent behavior of the IRPs may be explained, at
least partially, as follows. The cubane ISC of IRP1, which converts the
protein from an RNA-binding protein to a cytosolic aconitase, is
oxidation-sensitive and susceptible to degradation by oxidants
including oxygen, superoxide, hydrogen peroxide, nitric oxide, and
peroxynitrite [230–234]. Thus, such conditions may lead to an
increase in the IRE-binding pool of IRP1. With respect to the role of
ascorbate in modulating cellular iron metabolism, ascorbate may be
expected to play a more pronounced role in enhancing the IRP1/
aconitase switch mechanism described above, owing to the increased
levels of the RNA-binding form of the protein, under the higher
oxygen levels found under standard cell culture conditions [171].
In the case of IRP2, the high oxygen concentrations experienced
under typical cell culture conditions are thought to stabilize the
di-iron center in the hemerythrin domain of the FBXL5 and
thereby downregulate IRP2 levels by the activity of the FBXL5-
containing ubiquitin E3 ligase complex [201–204]. In contrast, the
degradation of IRP2 in response to iron proceeds efficiently under
mildly hypoxic conditions typical of the in vivo environment (viz.
3% oxygen), but this is compromised under conditions of severe
hypoxia (viz. 0.1% oxygen) [209]. Additionally, IRP2 is stabilized by
exposure to H2O2 [209]. Under such highly oxidizing conditions,
H2O2-dependent oxidation of Fe(II) to Fe(III) could occur, decreas-
ing the formation of the di-iron center of FBXL5 [209]. Intriguingly,
the protective effect of H2O2 on IRP2 protein stability can be
abolished by ascorbate [209], which is consistent with earlier
studies demonstrating that ascorbate promotes IRP2 degradation
[208] (see above). Taken together, these results may suggest that
under 21% oxygen, in which levels of oxidants such as H2O2 are
typically higher, ascorbate may play a proportionally greater role
in promoting protection and/or formation of the di-iron center of
FBXL5, leading to increased levels of FBXL5 and subsequent
downregulation of IRP2. Clearly, further studies are needed to
elucidate the various possibilities under different oxygen levels.
Second, HIF-dependent regulation of iron metabolism is also
known to be different in vivo compared to that found in cell culture
[219–221]. This differential regulation could be due to the effects
that hyperoxia has on PHD activity [215]. That is, PHD activity has
been shown to be increased under such conditions, leading to far
lower HIF1 levels than those typically observed under in vivo
oxygen levels [219–221]. Considering the possible difference in
effects of ascorbate on iron metabolism under 21% oxygen and
more hypoxic conditions, it may be expected that the effect of
ascorbate on downregulating HIF in a PHD-dependent manner
would be more pronounced at lower oxygen concentrations, which
are typical of the in vivo situation. Indeed, this is precisely what is
observed in experimental studies [219–221].
Such potential differences in the role of ascorbate in regulating
iron metabolism under hyperoxic cell culture conditions and
mildly hypoxic in vivo conditions should be taken into considera-
tion in further studies in this area.
Conclusion
In this review, we have explored the emerging hypothesis that Asc
contributes to cellular physiology, in part by functioning as a mod-
ulator of cellular iron metabolism. Indeed, accumulating evidence
 D.J.R. Lane, D.R. Richardson / Free Radical Biology and Medicine 75 (2014) 69–83
strongly suggests that in addition to the known ability of dietary Asc
to enhance nonheme iron absorption in the gut, Asc can regulate
cellular iron uptake and downstream cellular metabolism. Vitamin C
regulates iron metabolism by increasing Tf-dependent and non-Tf iron
uptake, the latter of which occurs by a novel transplasma membrane
ascorbate-cycling mechanism, stimulating ferritin synthesis, inhibiting
lysosomal ferritin degradation via autophagy, and decreasing cellular
iron efflux.
Recent evidence indicates that Asc stimulates iron uptake by
the classical Tf–iron uptake pathway, which provides almost all
iron for cellular demands and erythropoiesis under physiological
conditions. Ascorbate acts to enhance Tf-dependent iron uptake by
an intracellular reductive mechanism, strongly suggesting that it
may act to stimulate iron mobilization from the endosome into the
cell. The ability of Asc to regulate Tf–iron uptake could help
explain the metabolic defect that contributes to Asc-deficiency-
induced anemia.
Additionally, Asc appears to be capable of regulating both the
IRP–IRE and the HIF systems, which are integrally involved in
cellular and systemic iron homeostasis, although some crucial
mechanistic details remain the subject of controversy. However,
there is little doubt that Asc plays an active role in regulating these
processes, and the case for the multifaceted involvement of
ascorbate in iron metabolism is strong. As such, future studies
should aim to directly assess the mechanisms of involvement of
Asc in all tiers of mammalian iron homeostasis.
Acknowledgments
D.J.R.L. thanks the Cancer Institute New South Wales for an
Early Career Fellowship (10/ECF/2-18) and the National Health and
Medical Research Council (NHMRC) of Australia for an Early Career
Postdoctoral Fellowship (1013810). D.R.R. thanks the NHMRC for a
Senior Principal Research Fellowship and project grants.
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