Abnormal Iron Metabolism and Its Effect on Dentistry

2.1 Iron uptake

Owing to certain specific mechanisms (as explained inFigure 1): (1) transport mechanisms were not required for iron absorption until relatively late in evolution when the environment became oxidising and iron became insoluble, and (2) a range of sources can function as iron providers, bacterial iron assimilation happens via a variety of routes. Many bacteria, in addition, have numerous iron absorption mechanisms. This allows them to acquire iron from a variety of settings and sources. Bacteria can get iron from a number of sources, but regardless of where it comes from, it must be delivered to the cytoplasm through numerous microbial surface layers. An outside membrane, a peptidoglycan layer, and an innermost intracellular membrane are the minimum layers for Gram-negative bacteria. The periplasm, or gap between the outer and inner membranes, is where the peptidoglycan cell wall is found. Gram-positive cells, on the other hand, may only have an exterior peptidoglycan cell wall that is thick and strongly cross-linked. On the basis of the iron source and the manner in which iron is mobilised, a wide range of iron transport systems may be differentiated, although they all follow a similar pattern. Passage across the outer membrane for iron complexed to a carrier requires the presence of an outer membrane receptor protein with a syntactic domain identical to that of the iron complexed to a carrier and is iron-controlled. A receptor protein is specialised for and binds to a certain iron-carrier complex, and it is occasionally generated in large quantities only when that iron complex is accessible [5].

Second, the cytoplasmic membrane proteins TonB, ExbB and ExbD are required for iron entry into the periplasm, whether it is complexed or free. Members of the ABC super transporter family are also engaged in cytoplasmic membrane transport. The transport components, in this case, include a peripheral cytoplasmic membrane, ATPase with two copies and a distinct ATP-binding site motif, as well as two hydrophobic cytoplasmic membrane proteins. In summary, an outer membrane receptor protein, a TonB system and an ABC transporter are required for iron entrance into the cytoplasm of Gram-negative bacteria. The proton-motive force and ATP, respectively, are required for passage across the outer and cytoplasmic membranes. TonB systems have broad specificity, whereas ABC transporters recognise several iron complexes if they are physically related. Outer membrane receptor proteins bind just one particular iron complex, whereas TonB systems have broad specificity [6].

The synthesis and secretion of tiny (600–1000 Da) iron-chelating molecules known as siderophores is a significant method by which bacteria acquire iron. Siderophores are made up of ordinary amino acids, nonprotein amino acids, hydroxy acids, and their production does not need ribosomes despite the presence of amide bonds. Instead, a thiotemplate technique is used, which is quite similar to the one used to make some peptide antibiotics. The mechanism of iron release from siderophores is unknown. Free siderophores, or modified forms, are discharged into the medium when ferrisiderophores enter the cytoplasm. Enzymatic reduction of iron is considered to be the release mechanism since siderophores: (1) bind Fe(II) less readily than Fe(III); and (2) the cytoplasm is a reducing environment [7].

Microbes that can live in oxygen-depleted habitats, such as swamps, intestines and marshes, or acidic environments, where reduced iron is stable and soluble, benefit from the ferrous iron transfer. Fe(II) may enter the periplasm through holes in the outer membrane, and bacteria can transport it through the inner membrane through a number of mechanisms. Some of these cytoplasmic membrane transporters have a broad transition metal selectivity but just a weak affinity for ferrous iron. There are, however, systems that exclusively work with Fe(II) as a substrate. The feo operon encodes one such mechanism that is important in certain bacteria (feoABC) [8]. Members of the OFeT (oxidase-dependent iron transporter) family, which were initially discovered in lower eukaryotes, are another widely dispersed group of Fe(II) transporter proteins. Finally, certain aerotolerant bacteria, such as the Gram-positive Streptococcus mutans, acquire iron by converting surface-bound Fe(III) to Fe via a reductase that is exposed on the cell surface (II). The iron is subsequently delivered to the cytoplasm by a ferrous ion transporter. The ABC type of ferric iron acquisition mechanism is found in a variety of Gram-negative taxa, including Serratia, where it was identified and named Sfu type transport. Fe(III) is accepted by a periplasmic binding protein, which then delivers it to the transporter’s cytoplasmic components, which internalise the iron. Uptake systems with outer-membrane components can also work in tandem with ferric iron transporters [9].

The bulk of iron in animals is found intracellularly in the form of heme (Hm). Hm, in turn, is a prosthetic group of proteins that includes haemoglobin (Hb), myoglobin and Hm-containing proteins like cytochromes. Iron assimilation routes that detect free Hm are similar to those that identify iron–siderophore complexes; they need (1) a TonB-dependent outer-membrane receptor protein; and (2) an ABC transporter for cytoplasmic membrane crossing [10]. Hm can be removed from Hb by a variety of genera. TonB-dependent Hb-binding proteins are found in the outer membranes of Neisseria and Haemophilus spp. Surprisingly, both of these taxa contain additional TonB-dependent receptors that let them get iron from Hb–Hp complexes. These Hb–Hp receptors might be made up of two distinct proteins. Serratia marcescens has a unique mechanism for the first steps in getting iron from Hm or Hb. This bacterium secretes a tiny protein (HasA) that acts as a hemophore via an ABC transporter (Hbp). Only Haemophilus strains have been shown to use Hm in conjunction with hemopexin. The mechanism is not fully understood, but it appears that three genes are necessary, one of which appears to encode a big secreted Hbp (HxuA). HxuA binds hemopexin, removes it, and transports it to an outer-membrane receptor [11].

Iron trafficking exemplifies the cycle economy. During erythrocyte phagocytosis, the majority of iron (20–25 mg/day) is recycled by macrophages; only 1–2 mg of iron is absorbed daily in the stomach, compensating for a loss of the same amount (Figure 2) [13]. The duodenum is the location of controlled non-heme iron uptake; nonheme iron is imported from the lumen via the apical divalent metal transporter 1 after duodenal cytochrome B reductase converts ferric to ferrous iron (DCYTBH) (DMT1). There are no known mechanisms by which heme iron absorbs more than non-heme iron. Non-utilised iron in enterocytes is either retained in ferritin (and lost by mucosal shedding) or exported to plasma through basolateral membrane ferroportin (and lost with mucosal shedding) [14].

Iron availability influences the expression of genes that code for proteins required for high-affinity iron absorption. Fur is a crucial regulatory protein found in most Gram-negative and Gram-positive bacteria with low GC content DNA. Fur is an Apo-repressor, a short histidine-rich polypeptide that binds DNA in the presence of its corepressor Fe(II). Fur’s negative regulation of genes does not fully explain iron’s regulatory actions. Although Fur represses most iron-regulated genes under iron-rich environments, some are positively controlled by Fur, and others are only activated by iron in the absence of Fur (Figure 3) [15].

2.2 Iron distribution

Transferrin binds to iron in the bloodstream and distributes it to storage and use sites. Only 30–40% of transferrin’s iron-binding capacity is used in ordinary physiological circumstances; hence, transferrin-bound iron is only w4 mg, yet it is the most significant dynamic iron pool. Transferrin-bound iron penetrates target cells, predominantly erythroid cells, but also immune and hepatic cells, via a highly specialised method of receptor-mediated endocytosis (Figure 1). Patches of cell-surface membrane bearing receptor–ligand complexes invaginate to create clathrin-coated endosomes as distinct transferrin binds to transferrin receptor 1 (TfR1) at the plasma membrane (siderosomes) [16]. A ferrireductase reduces Fe3+ to Fe2+, which is subsequently transferred to the cytoplasm by DMT1, while TfR1 is recycled to the cell membrane and transferrin is lost. Mitoferrin, a mitochondrial iron importer, is important in providing iron to ferrochelatase for insertion into protoporphyrin IX and to produce haem (the penultimate step of mitochondrial haem production) within the erythroblast (Figure 1). There are some indications that iron might be transported straight from the siderosomes to the mitochondria in growing erythroid cells. Finally, haem exporters transport haem from mitochondria to cytosol and eliminate excess haem from erythroid cells (Figure 1) [16].

2.3 Iron storage

As senescent erythrocytes are phagocytosed by RES macrophages, haemoglobin iron turnover is high. Haem is metabolised by haem oxygenase within the phagocytic vesicles, and the liberated Fe2+ is transported to the cytoplasm by NRAMP1 (natural resistance-associated macrophage protein-1), a transport protein related to DMT1 (Figure 1). Macrophages may also acquire iron from bacteria and apoptotic cells, as well as from plasma via the actions of DMT1 and TfR1 (Figure 1) [17]. Iron may be stored in the cells in two ways: ferritin in the cytosol and haemosiderin in the lysosomes when ferritin is broken down. Haemosiderin is found in just a small percentage of normal human iron reserves, primarily in macrophages, but it rises substantially when the body is overloaded with iron. Iron storage in macrophages is also safe since it does not cause oxidative damage. Ferroportin 1, the same iron-export protein found in the duodenal enterocyte, and caeruloplasmin2 are largely responsible for iron export from macrophages to transferrin (Figure 1) [18]. Macrophage iron recycling provides the majority of the iron necessary for the daily synthesis of 300 billion red blood cells (20–30 mg). While a result, internal iron turnover is required to satisfy the bone marrow needs for erythropoiesis, as daily absorption (1–2 mg) only balances daily loss. 1–3 The liver is the other major iron storage organ, and the production of free radicals and lipid peroxidation products in iron-overload conditions can lead to hepatic tissue damage, cirrhosis, and hepatocellular cancer [19]. TfR1 and TfR2 mediate the liver’s absorption of transferrin-bound iron from plasma (Figure 1), however, it can also get iron from non-transferrin-bound iron (through a carrier-mediated mechanism similar to DMT1), ferritin, haemoglobine–haptoglobin complexes, and haeme–haemopexin complexes. Ferroportin 1 is thought to be the sole protein that mediates the export of iron from hepatocytes, which is then oxidised by caeruloplasmin and attached to transferrin2 (Figure 1). Heart failure is the primary cause of death in individuals with untreated hereditary haemochromatosis or transfusion-associated iron overload, thus iron storage in cardiomyocytes is of significant interest. Excess iron in cardiac cells can cause oxidative stress and impair myocardial function owing to DNA damage caused by hydrogen peroxide via the Fenton reaction [20].

2.4 Regulation of iron homoeostasis

Body iron reserves, hypoxia, inflammation and erythropoiesis rate all influence iron absorption by duodenal enterocytes. The crypt programming model and the hepcidin model are two regulatory models that have been presented as potential contributors to iron absorption control [21].

Enterocytes in the crypts of the duodenum take up iron from the plasma via TfR1 and TfR2, according to the crypt programming hypothesis. The interaction of cytosolic iron regulatory proteins (IRPs) 1 and 2 with iron-responsive elements is controlled by intracellular iron content (IREs). IRP1 binds to the IREs of TfR1, DMT1, and ferroportin 1 mRNA in the absence of iron, stabilising the transcript, allowing translation to occur and the proteins to be synthesised. As a result, increased IRP-binding activity indicates low body iron reserves, which leads to overexpression of these proteins in the duodenum, boosting dietary iron absorption. When IRPs attach to ferritin mRNA’s IREs, the transcript’s translation is interrupted and synthesis is halted. As a result, ferritin concentrations are inversely controlled, increasing in iron-rich states and decreasing in iron-deficient conditions [22].

The hepcidin model proposes that hepcidin is produced mainly by hepatocytes in response to the iron content of the blood. Then, hepcidin is secreted into the bloodstream and interacts with villous enterocytes to regulate the rate of iron absorption by controlling the expression of ferroportin 1 at their basolateral membranes. The binding of hepcidin to ferroportin 1 initially causes Janus kinase 2-mediated tyrosine phosphorylation of the cytosolic loop of the carrier protein, phosphorylated ferroportin 1 is then internalised, dephosphorylated, ubiquitinated and ultimately degraded in the late endosome/lysosome compartment. Ferroportin 1 molecules, present in macrophages and liver, also targets for hepcidin [23].

The sensing process most likely includes local iron-induced synthesis of bone morphogenic proteins (BMPs) such as BMP6 within normal iron concentration limits. BMP6 interacts with hepatocyte cell surface BMP receptors (BMPRs) I and II, as well as the BMP coreceptor, haemojuvelin (HJV), triggering an intracellular signal by phosphorylation of small mothers against decapentaplegic (Smad) proteins. Before translocating to the nucleus and triggering hepcidin expression14, phosphorylated Smad1, Smad5 and Smad8 form a complex with the shared mediator Smad4 (Figure 2). The soluble form of HJV (sHJV), whose release (HJV shedding) is prevented by rising extracellular iron concentrations, is thought to compete with its membrane-anchored counterpart for BMPR binding, resulting in iron-sensitive hepcidin expression16 (Figure 2). Other mediators and modulators, including Smad6 and Smad7, may be stimulated by iron, and these mediators and modulators appear to dampen the signal for hepcidin activation (Figure 2) [24].

2.5 Effects of inflammation on iron homoeostasis and erythropoiesis

Cancer, rheumatoid arthritis, inflammatory bowel disease, congestive heart failure, sepsis and chronic renal failure are all known to induce persistent inflammation. This anaemia might be caused by the underlying process activating the immune system, as well as immunological and inflammatory cytokines such as tumour necrosis factor alpha (TNFa), interferon-gamma (IFNg), interleukins (IL) 1, 6, 8, and 10. Several pathophysiological processes (cytokines) may be implicated in anaemia of chronic disease (ACD) (Figure 3) [25]:

1. Dyserythropoiesis, red blood cell destruction, and increased erythrophagocytosis cause a reduction in red blood cell half-life (TNFa).

2. Inadequate EPO responses for the degree of anaemia in most, but not all, patients, such as those with juvenile chronic arthritis with systemic start (IL-1 and TNFa).

3. Erythroid cell response to EPO is impaired (IFNg, IL-1, TNFa, hepcidin).

4. Erythroid cell growth and differentiation are slowed (IFNg, IL-1, TNFa, and a1-antitrypsin).

5. Pathological iron homoeostasis caused by increased DMT1 (IFNg) and TfR (IL-10) expression in macrophages, decreased ferroportin 1 expression in enterocytes (inhibition of iron absorption) and macrophages (inhibition of iron recirculation), and increased ferritin production ‘(TNFa, IL-1, IL-6, IL-10) (increased iron storage) Inflammatory cytokines like IL-6’ activate Janus kinases, which phosphorylate Stat3 and activate it, which upregulate hepcidin transcription. Stat3 translocation to the nucleus and binding to the Stat response element in the proximal promoter of the hepcidin gene leads to enhanced hepcidin release. This element appears to be controlled by Smad activation, which is necessary for complete promoter activity, via the adjacent BMP-responsive element. The SmadeStat complex, which puts the distal and proximal areas of the hepcidin promoter into physical contact, is hypothesised to interact with a distal BMP responsive element location. As a result, it appears that Smad signalling is critical for the appropriate staging of the inflammatory response. Stat3 activation has also been demonstrated to modulate hepcidin levels without producing inflammation (for example, people with glycogen storage disease type 1a who had hepatic adenomas overexpressed hepcidin due to Stat3 activation)46 (Figure 2). Stress mechanisms signalling through the cellular endoplasmic reticulum unfolded protein response have also been shown to stimulate hepcidin production. The hepatic acute-phase response to LPS, IL-6 and IL-1b has been related to the unfolded protein response, suggesting that hepcidin gene expression may be regulated by another layer of endogenous regulation during inflammation (Figure 2). Low blood iron and reduced transferrin saturation are produced by iron diversion to the RES (functional iron deficit, FID), iron-restricted erythropoiesis, and mild-to-moderate anaemia, despite normal or high serum ferritin levels [26].

3.1 Iron deficiency

In the human body, there is a balance between iron absorption, iron transit and iron storage under physiological circumstances. ID and iron deficiency anaemia (IDA) can be caused by a combination of three risk factors: higher iron needs, restricted external supply and increased blood loss [27]. There are two types of ID: absolute and functional. Iron reserves are reduced in absolute ID; in functional iron deficiency (FID), iron stores are full but cannot be mobilised as quickly as needed from the RES macrophages to the bone marrow. Diagnostic tests with values are given in Table 1.

3.2 Iron overload

Levels of Hepcidin are excessively lower-degree of overload of iron in idiopathic iron overload illness and primary haemochromatosis. This is due to mutations in the genes that code for ‘HFE (haemochromatosis type 1)’, ‘haemojuvelin (HJV; juvenile haemochromatosis 2a)’, and ‘transferrin receptor 2 (TfR2; haemochromatosis type 3)’; these mutations cause hepcidin synthesis to be dysregulated [32]. The only exceptions are mutations that disrupt hepcidin or ferroportin (juvenile haemochromatosis 2b) (haemochromatosis type 4). Low plasma hepcidin causes high ferroportin levels, allowing for greater iron absorption, hepatic iron overload and low iron levels in macrophages. In addition, non-transferrin bound iron emerges as transferrin gets saturated in iron-overload situations. A portion of this labile plasma iron is extremely reactive, resulting in the production of free radicals. Despite the fact that the HFE gene has at least 32 mutations, the most prevalent form of haemochromatosis type 1 is caused by the missense Cys282Tyr mutation. Haemochromatosis type 1 is a disease with a wide range of penetrance and heterogeneity, although the Cys282Tyr mutation is found in the great majority of people with the disorder. Because the Cys282Tyr mutant HFE protein is unable to bind b2 microglobulin, it does not reach the cell membrane, resulting in a misfolded, non-functional protein. Iron overload can be caused by mutations in the ferroportin gene (haemochromatosis type 4) that result in the loss of iron-export capacity, hyperferritinaemia with no increase in transferrin saturation, and macrophage iron overload, or a loss of hepcidin-binding activity, which has been linked to iron overload. Plasma hepcidin levels rise in cases of secondary iron overload-induced by persistent transfusion treatment (e.g. severe thalassemia, aplastic anaemia, etc.), prompting ferroportin breakdown. Increased amounts of diferric transferrin, which are elevated in iron overload, promote TfR2 expression at the hepatocyte membrane. When diferric transferrin binds to TfR2, HJV cleavage by furin is blocked, inhibiting the release of soluble HJV and resulting in enhanced cell-surface HJV-mediated response to BMPs and higher hepcidin levels. Iron absorption from the stomach is restricted, macrophage export is inhibited, and iron storage is increased when ferroportin levels are low [33].

3.3 Assessment of defective iron metabolism

4.1 Iron deficiency anaemia

The most prevalent kind of anaemia is iron deficiency anaemia (IDA), which affects more women than males. Due to persistent blood loss associated with heavy menstrual flow, it is estimated that 20% of women of reproductive age in the United States are iron deficient. Furthermore, 2% of adult males are iron deficient due to persistent blood loss caused by gastrointestinal illnesses including peptic ulcer, diverticulosis, or cancer [37].

4.2 Iron overload

Hemochromatosis is the abnormal accumulation of iron in parenchymal organs, leading to organ toxicity. It is the most common inherited liver disease in whites and the most common autosomal recessive genetic disorder. Genetic haemochromatosis (GH), which is related to the HFE gene p.Cys282Tyr mutation, is the most common form of inherited iron overload disease in European population descendants.

4.2.1 Symptoms

The classic tetrad of manifestations resulting from hemochromatosis consists of: (1) cirrhosis, (2) diabetes mellitus, (3) hyperpigmentation of the skin and teeth, and (4) cardiac failure. Clinical consequences also include hepatocellular carcinoma, impotence and arthritis (Figures 4 and 5) [9].

Symptoms can vary from burning mouth syndrome to bald and inflamed tongue [9].

4.2.2 Pathophysiology

Periodontitis is linked to an inflammatory response triggered by changes in the subgingival biofilm. Inflammation causes iron sequestration inside macrophages in healthy people, depriving bacteria of iron. Iron bioavailability in biological fluids, particularly those of the oral cavity, is enhanced in GH patients with excessively high TSAT, resulting in an increased risk of severe periodontitis. The existence of iron deposits in oral tissues of haemochromatosis patients has also been documented in the literature (Figure 6). The majority of people with haemochromatosis are now asymptomatic, and the skin and mucosal colouration caused by iron deposits have improved dramatically. The occurrence of asymptomatic iron deposits in oral tissues, however, cannot be ruled out [10, 11].

Iron is connected with transferrin in plasma, which increases its bioavailability for cells. The ratio between the total number of iron-binding sites on patient plasma transferrin and the number of binding sites occupied by iron is known as transferrin saturation (TSAT). TSAT is normally seen in the range of 20% to 45 per cent. Hepcidin regulates systemic iron metabolism, and its expression level is tuned to TSAT to regulate plasma iron levels. Hepcidin insufficiency is a symptom of GH, which is caused by a change in the HFE-linked transduction signalling pathway. TSAT levels rise as a result of the iron outflow from macrophages and enterocytes. Non-transferrin-bound iron (NTBI), an aberrant biochemical type of iron, arises in the plasma when TSAT surpasses 45 per cent. The liver and heart are particularly vulnerable to NTBI, which explains why the typical type of GH causes hepatic cirrhosis and diabetes. However, in the absence of cirrhosis or diabetes, the majority of GH patients remain asymptomatic or have chronic tiredness, abnormal serum transaminase levels, rheumatism, and osteoporosis. Cells manufacture ferritin to store excess iron in order to avoid iron toxicity. As a result, the tissue iron reserves are reflected in plasma ferritin levels. The standard treatment is phlebotomy therapy, which is used to take out excess iron and then prevent it from being reconstituted. The gold standard for both initial treatment and maintenance therapy, according to the leading international standards, is serum ferritin levels of less than 50 g/L [13].