Haemochromatosis: Pathophysiology and the red blood cell 1

Abstract

Haemochromatosis remains the most prevalent genetic disorder of Caucasian populations in Australia and the United States, occurring in ∼1 of 200 individuals and having a carrier frequency of 10–14%. Hereditary haemochromatosis is an autosomal recessive condition, that is phenotypically characterised by a gradual accumulation of iron, above and beyond that required for biological function. Once the binding capacity of iron carriers reaches saturation, the highly reactive free iron generates radicals that may lead to widespread cellular dysfunction. Thus, the compounding effects of systemic iron overload and the associated oxidative stress in untreated haemochromatosis patients results in tissue damage precipitating severe complications, including: liver cirrhosis, hepatocellular cancer, cardiomyopathy, and diabetes. The primary treatment indicated for individuals with haemochromatosis is venesection therapy (i.e., regular bloodletting of ∼450 mL). Given the frequency of venesection required to decrease and normalise the elevated iron levels, this population may serve as a valuable source of blood products which are in short supply. While the complications associated with elevated iron deposits are frequently reported, the influence of haemochromatosis on the rheological properties of blood and red blood cells (RBC) – major determinants of microvascular blood flow and tissue perfusion – are poorly understood. Limited studies investigating haemorheology in patients with haemochromatosis have reported altered physical properties of blood, which may partly explain the comorbidities associated with the disorder. The current review will explore the aetiology, pathology, and clinical implications of haemochromatosis disease and the associated oxidative stress, with particular emphasis on RBC.

Keywords

Hemorheology iron overload oxidative stress

1 Iron homeostasis

The capacity of iron to generate free radicals and participate in redox reactions makes for an extremely versatile element which is involved in a variety of physiological processes vital for healthy cellular function. Within the body, iron has three primary functions: i. to form proton gradients to facilitate chemiosmosis in the electron transport chain, imperative for cellular respiration and energy liberation; ii. to generate destructive free radicals for immune regulation, assisting macrophage phagocytosis and immune defence; and, iii. to form metalloproteins such as the haem complex within haemoglobin, vital for the binding and transport of oxygen [1]. Given the importance of the iron for biological processes, and that dietary intake is limited, iron levels must be strictly regulated within the body (Fig. 1). In healthy adult males, ∼10% of iron is found within myoglobin and iron-based enzymes, ∼10–30% is found within ferritin and transferrin, and the remaining ∼65% is located within haemoglobin [2]. As red blood cells (RBC) have a high turnover rate, with a lifespan of ∼120 d, the largest fluctuations in systemic iron content occurs through the RBC lifecycle: old RBC are removed from circulation, haemoglobin is recovered by macrophages, and iron is recycled for continual erythropoiesis [3].

Fig.1

Iron cycle in humans. The rate of iron absorption and iron loss is well balanced (∼1-2 mg/d), with much of the iron contained within RBC being recaptured to minimise excretion.

2 Iron absorption, transport and storage

Hepcidin is the key hepatic hormone involved in the gastrointestinal regulation of iron absorption [4]. Induced by elevated iron stores and inflammation, hepcidin results in the inhibition of the duodenal iron transporter ferroportin, consequently decreasing iron absorption through enterocytes (Fig. 2) [5, 6]. Despite elevated iron stores, the low hepcidin concentration observed in haemochromatosis disease (suggesting malfunctioning upregulation) [7, 8], results in a paradoxical increase in iron uptake via ferroportin transporters and subsequent increases in plasma iron concentrations [9].

Fig.2

Iron transport through duodenal enterocytes. Following conversion from Fe^3 + to Fe^2 + via ferrireductase (cytochrome b561) [10], the DMT1 transporter located on the apical membrane allows non-haem iron transport from the gastrointestinal lumen, into the enterocyte [11]. Conversely, the HCP1 transporter allows haem-based iron to enter the enterocyte [12]. In the absence of hepcidin, the ferroportin transporter of the basolateral membrane remains active and allows Fe^2 + to pass through the enterocyte, where it is converted back to Fe^3 + by the hephaestin enzyme [13]. In the presence of elevated hepcidin concentrations, degradation of ferroportin occurs, resulting in a decreased iron transport [14].

Given iron is vital for physiological functioning, a mismatch between the absorption and excretion of iron propagates numerous pathologies. Deficiency in iron absorption, or elevated loss of iron, for example leads to iron deficiency anaemia and poor oxygen delivery. Conversely, overexpression of the protein channels that absorb iron, or a dysfunction of the iron-regulatory hormone hepcidin, results in excessive accumulation of iron. While increased iron absorption is initially buffered by transferrin, once the binding capacity of transferrin is saturated, plasma iron concentration increases [15, 16]. Due to the reactive nature of iron, when iron is in its ferrous (Fe^2 +) state it may oxidise and damage surrounding cells; however, the body can safely transport and store excess iron in a more stable, less reactive, ferric (Fe^3 +) form created by the binding of ferrous iron to the protein complexes transferrin (for systemic iron transport) and apoferritin to produce ferritin (for iron storage – predominantly in hepatocytes). While stored iron located within the ferritin complex is a soluble molecule with a protective outer protein shell, elevated concentrations of iron can denature ferritin, converting it to haemosiderin – containing non-soluble forms of iron. Haemosiderin, like ferritin, may also be stored within tissues; however, unlike ferritin, haemosiderin is associated with organ damage, due to an increased ratio of iron– to– protein concentration and an irregular dispersion of electron-dense particles with large oxidative capacities [17].

3 Diagnosis and genetic basis of haemochromatosis

Iron accumulation in haemochromatosis patients is a slow and progressive process, thus diagnosis and clinical manifestation generally occurs in the 4th to 5th decades of life [18, 19]. Common signs and symptoms include: arthralgia, fatigue, cardiac arrhythmias, abdominal pain and skin pigmentation [20, 21]. Following the onset of symptoms, haemochromatosis is initially identified by assessing transferrin saturation and ferritin levels; transferrin saturation >45% and elevated serum ferritin concentrations (>200 ng/mL) indicate haemochromatosis [22]. Following this, genetic testing can confirm the specific sub-type of the disorder [23].

Currently, there are five known genetic mutations associated with haemochromatosis. The aetiologies, inheritance and prevalence of these types of haemochromatosis are summarised in Table 1. The most prevalent mutation, resulting in type 1 (hereditary) haemochromatosis, occurs at the HFE gene on chromosome 6, substituting cysteine for tyrosine on codon 282 (HFE-C282Y) [24, 25]. In addition, a substitution of histidine for aspartic acid on codon 63 (HFE-H63D) is suggested to be a further cause of type 1 haemochromatosis, albeit at a much lower phenotypic penetrance [26]. HFE type 2A (juvenile) haemochromatosis occurs much less frequently than type 1, and is predominately due to a substitution of glycine for valine at codon 320 (G320V) of the haemojuvelin (HJV) gene on chromosome 1 [27, 28]. While most juvenile haemochromatosis cases are caused by mutations to the HJV gene, a small number of cases (referred to as type 2B haemochromatosis) are induced by a mutation in the HAMP gene (crucial for hepcidin expression), with substitution of cysteine for arginine at codon 70 or cysteine for tyrosine at codon 78, on chromosome 19 [29]. This mutation observed in type 2B haemochromatosis results in the inactivation of hepcidin [29]. An additional, less common form of iron overload is type 3 haemochromatosis, which is associated with mutations of the transferrin receptor 2 gene (TFR2) on chromosome 7 [30, 31]. The final two known causes of haemochromatosis, type 4 and type 5, are rare. These variants of the disorder are caused by mutations in the SLC40A1 and H-ferritin genes, respectively [32, 33].

Table 1
Phenotypic expression of haemochromatosis

Genetic Type Affected Gene Affected Protein Dysfunction Mode of Inheritance Reference

Haemochromatosis Type 1 (hereditary haemochromatosis) HFE HFE Low hepcidin concentration Autosomal recessive [24, 34]

Haemochromatosis Type 2A (juvenile haemochromatosis) HJV Haemojuvelin Ineffective hepcidin release Autosomal recessive [28]

Haemochromatosis Type 2B HAMP Hepcidin Low hepcidin concentration Autosomal recessive [29]

Haemochromatosis Type 3 TfR2 Transferrin Receptor 2 Low hepcidin concentration Autosomal recessive [30 , 36]

Haemochromatosis Type 4 SLC40A1 Ferroportin Dysregulation of ferroportin Autosomal dominant [32]

Haemochromatosis Type 5 H-Ferritin Ferritin Dysregulation of ferroportin Autosomal dominant [33]

Genetic Type	Affected Gene	Affected Protein	Dysfunction	Mode of Inheritance	Reference
Haemochromatosis Type 1 (hereditary haemochromatosis)	HFE	HFE	Low hepcidin concentration	Autosomal recessive	[24, 34]
Haemochromatosis Type 2A (juvenile haemochromatosis)	HJV	Haemojuvelin	Ineffective hepcidin release	Autosomal recessive	[28]
Haemochromatosis Type 2B	HAMP	Hepcidin	Low hepcidin concentration	Autosomal recessive	[29]
Haemochromatosis Type 3	TfR2	Transferrin Receptor 2	Low hepcidin concentration	Autosomal recessive	[30 , 36]
Haemochromatosis Type 4	SLC40A1	Ferroportin	Dysregulation of ferroportin	Autosomal dominant	[32]
Haemochromatosis Type 5	H-Ferritin	Ferritin	Dysregulation of ferroportin	Autosomal dominant	[33]

4 Clinical presentations

Given the reactivity of ferrous iron, numerous comorbidities are associated with uncontrolled haemochromatosis. In a cohort of 410 individuals with haemochromatosis, Adams et al. [20] observed that liver cirrhosis, arthritis, diabetes, and cardiac disease were common comorbidities. Niederau et al. [37] examined a cohort of 251 haemochromatosis patients for an average of 14 years, and reported that death by liver cancer was 119 times more frequent, compared with healthy individuals. Additionally, cardiomyopathy and diabetes mellitus were 14 times more likely to be the cause of death in those with haemochromatosis. Due to the severity of the comorbidities associated with haemochromatosis, early and optimal treatment is imperative.

5 Treatment of haemochromatosis

Given the incurable nature and life-threatening complications associated with uncontrolled haemochromatosis, treatment is vital. Treatment options principally involve one of two approaches: i. venesection therapy or ii. iron chelation pharmacotherapy. Venesection therapy is generally the primary treatment indicated for the majority of haemochromatosis populations, largely due to cost effectiveness and reduced side effects when compared with pharmacological interventions [38]. Venesection therapy is typically initiated when serum ferritin concentration surpasses 300μg/L (male) or 200μg/L (female), and ceases when these levels fall below 50μg/L [39]. Each venesection involves the removal of approximately 7 mL of blood per kilogram of body mass, which removes ∼200–250 mg of blood-based iron (i.e., ferritin and haemoglobin within RBC) [40, 41]. Current guidelines recommend a two-phase approach to venesection therapy. In order to drastically decrease body iron levels, the initial “induction phase” involves monthly repeats of venesection therapy, with regular monitoring every second treatment for haemoglobin and iron concentration [41]. Once iron concentration is reduced to target levels (i.e., 50μg/L), a “maintenance phase” is implemented. During this phase, a patient receives venesection therapy every 1–4 months to maintain desirable iron levels [41]. Patient compliance to venesection therapy is generally high during maintenance, with 84% of patients adhering to their treatment regime over the first 12 months [42].

The alternative treatment to venesection therapy involves pharmacological intervention with agents that chelate iron. This therapy is indicated for individuals who cannot support frequent blood donation, e.g., patients with heart failure and/or anaemia, or patients suffering acute iron toxicity [43]. Three common iron chelators for clinical use are deferoxamine, deferiprone and deferasirox. Deferoxamine is delivered subcutaneously at doses of 40 mg/kg of body mass, and increases mobilisation of iron deposited in parenchymal cells and macrophages [43]. Deferiprone is administered orally at of 75 mg/kg of body mass, and improves iron status through decreasing iron saturation within transferrin molecules [44]. Deferasirox is also administered orally (20 mg/kg), increasing iron mobilisation within cardiac myocytes [43]. While the aforementioned iron chelator agents are effective at decreasing plasma iron concentration, recent research investigating a novel iron chelator has shown that DIBI (a hydroxypyridinone-class of chelator) is effective at decreasing leukocyte recruitment whilst improving functional capillary density (a biomarker of the severity of inflammation) in endotoxemic mice [45]. Thus, due to its anti-inflammatory effects, DIBI may be useful in treating diseases characterised by high levels of iron and inflammatory markers, such as haemochromatosis [46].

6 Blood rheology in haemochromatosis

Blood is a non-Newtonian shear-thinning fluid, whereby its apparent viscosity is dependent on magnitudes of applied shear (for review, see Simmonds et al. [47]). Shear-thinning of blood is predominantly attributed to the cellular components of blood, particularly RBC [48]. The physical properties of RBC influence the rheological behaviour of blood through RBC aggregation in low-shear environments, and RBC deformability in high-shear flow. Alterations and/or damage to RBC are likely to propagate impaired blood flow, particularly through microvascular regions [49, 50], further facilitating the development of secondary complications and microvascular disease.

The ability of RBC to deform, and subsequently align in the streamlines of blood flow, is largely dependent on: i. a large surface-area-to-volume ratio of the cell; ii. the cytosolic viscosity of the cell – determined by intracellular haemoglobin concentration; and, iii. the elasticity of the cytoskeletal membrane [48]. Moreover, the “tank treading” motion of RBC is believed to be a key determinant of RBC orientation in shear flow, thereby increasing blood fluidity [51]. RBC deformability is important in facilitating orientation of RBC in high-velocity flow, and is required for RBC to traverse microcirculatory networks that are smaller than the cell itself [52]. Pretorius et al. [53] reported that the morphology of RBC from individuals with haemochromatosis deviates from the typical biconcave shape, instead presenting with irregular, elongated morphology. Further, Barton et al. [54] reported that RBC from haemochromatosis patients had elevated haemoglobin concentration and corpuscular volume. Given these changes in cell morphology and properties are also primary determinants of RBC deformability [47], it is not surprising that McNamee et al. [55] found that RBC deformability in haemochromatosis patients was significantly impaired compared with control. Their experimental investigation provided further insight, as the authors did not observe alteration in RBC deformability with acute ex vivo incubations of healthy RBC with iron, highlighting that rheological impairments are likely the result of chronic and systemic iron overload in haemochromatosis. The findings of Pretorius et al. [53] support this hypothesis, given their report that the altered RBC morphology in haemochromatosis could be normalised following blood incubation with iron chelators and antioxidants.

McNamee et al. [55] also investigated RBC aggregation in haemochromatosis patients, and found increased magnitudes and rates of aggregation relative to controls. When the haemochromatosis RBC were suspended in a plasma-free medium, however, the RBC aggregability of haemochromatosis was not different to control, suggesting that plasma factors likely determine the increased RBC aggregation in this disorder. The authors experimental investigation of acute iron incubation on healthy RBC did not significantly increase RBC aggregation or aggregability. Given RBC aggregation is the primary determinant of low-shear viscosity, increases in this behaviour, or failure of formed rouleaux to disaggregate may explain, in part, some of the comorbidities that present in uncontrolled haemochromatosis. The reversibility of the increased cell aggregation has not previously been investigated in this population; however, given plasma factors appear to explain the increased aggregation, perhaps the use of iron chelators and antioxidants may also normalise this parameter. The ability of venesection therapy to normalise the physical properties of RBC and the rheological properties of blood should be determined.

7 Free radical production in haemochromatosis

The mammalian body possesses a vast network of readily available antioxidant defence mechanisms including various enzymatic (e.g., superoxide dismutase) and non-enzymatic (e.g. Vitamin A, C, E) scavengers [56]. When production of oxidative species overcome the body’s capacity to buffer these reactions, oxidative stress occurs. In untreated haemochromatosis patients, for example, when iron homeostasis cannot be maintained, the excess iron facilitates production of free radicals, thereby quenching available antioxidants [57]. Specifically, within a cell’s mitochondrion, iron participates in a one-electron transfer reaction called the “Fenton Reaction” [58]. In the presence of hydrogen peroxide, ferrous iron catalyses the formation of highly reactive hydroxyl radicals, while increasing its own oxidation state (i.e., Fe^2 + → Fe^3 +) [58]. The formation of both hydroxyl radicals and ferric iron is associated with widespread damage to biological structures, including nucleic acids, cell membranes, and proteins [59]. In tandem, the reaction of ferric iron and superoxide occurs, providing the ferrous iron needed to form hydroxyl radicals. The collective reaction is referred to as the Haber-Weiss Cycle (Fig. 3).

Fig.3

Depiction of the Haber-Weiss Cycle. Initially, ferric iron reacts with superoxide (·O₂) resulting in oxygen and ferric iron formation. Ferric iron can subsequently react with hydrogen peroxide forming hydroxyl radicals, hydrogen peroxide and ferric iron.

Due to the involvement of iron in the production of oxidative species, the relationship between haemochromatosis and markers of oxidative stress is highly relevant. Earlier reports have demonstrated that haemochromatosis (HFE–C282Y) patients have a higher urinary concentration of 8-iso-PGF (an oxidative stress marker) when compared to healthy age-matched control groups [57]. Following iron-depletion therapy, however, urinary excretion of 8-iso-PGF in haemochromatosis patients was similar to that of healthy controls [57]. Given that the complications associated with haemochromatosis (such as cardiomyopathy and diabetes) have been suggested to be potentiated by oxidative stress [60], the importance of normalising iron status in individuals with haemochromatosis is paramount.

8 Influence of oxidative stress and iron overload on blood flow

Potentially exacerbated by the production of excess free radicals, the macro- and micro-vascular complications associated with haemochromatosis are suggestive of impaired blood rheology and endothelial function [61]. Given that increased iron content in haemochromatosis induces systemic oxidative stress, and the circulatory system traverses the entirety of the body, the constituents of blood and the endothelium may be susceptible to these damaging free radicals. Indeed, animal models of diabetes, a disease characterised by high indices of oxidative stress [62], have shown that vascular reactivity is impaired following exposure to high free radical concentrations [63]. Likewise, Gaenzer et al. [61] reported that untreated male haemochromatosis patients have significant decrements in endothelial-dependent vasodilation and significant increases in carotid intima-media thickness, when compared to age-matched male controls. Moreover, the authors reported that routine venesection therapy in haemochromatosis patients concurrently lowered plasma ferritin concentration whilst improving flow-mediated dilation (i.e., an index of endothelium-dependent vasoreactivity). Collectively, these data indicate that macro-vascular blood flow is impaired in haemochromatosis patients, thereby potentially hindering oxygen and nutrient delivery.

9 Potential mechanisms of altered blood rheology in haemochromatosis

The altered blood rheology in haemochromatosis is not well understood, although factors relating to the irregular morphology, intracellular viscosity, and iron-mediated free radical production in this disorder are likely contributors. It is plausible that the elongated and irregular morphology in haemochromatosis in the presence of plasma iron may induce the hyperaggregation observed in McNamee et al. [55], given that aggregate formation requires adequate surface area for cell-cell contact [64]. Moreover, the increased mean corpuscular haemoglobin concentration, and mean cell volume, reported in haemochromatosis may contribute to altered RBC deformability, given that cytosolic haemoglobin is the primary determinant of intracellular viscosity, and increased cell volume negatively impacts the cell surface area relative to volume, which is detrimental to cell deformability [48]. The primary effect that mediates impaired cell deformability appears to be, however, the likelihood for excess free radical production catalysed by iron overload, as discussed below.

While the direct influence of haemochromatosis on the physical properties of RBC is not fully understood, blood exposure to free radicals dramatically alters cell morphology which may be associated with impaired function [65, 66]. Specifically, free radicals alter RBC morphology by: degrading surface membrane proteins and inducing lipid peroxidation [67, 68]; quenching nitric oxide, limiting its availability [69]; and, facilitating spectrin-haemoglobin crosslinking and thus decreasing membrane elasticity [67]. These morphological changes collectively lead to impaired mechanical function of the RBC. Specific effects of the site – i.e., intra- or extracellular – of RBC exposure to free radicals appears to be important. Intracellular generation of superoxide (a potent free radical), for example, significantly decreases RBC deformability [65]. Moreover, intracellularly-generated superoxide was reported to increase the susceptibility of RBC to mechanical stress [70]. That is, RBC previously exposed to intracellular superoxide exhibited greater impairment in cell deformability following exposure to supraphysiological shear stress, when compared with untreated RBC. Exposure of RBC to extracellularly-generated superoxide, on the other hand, also impairs RBC deformability, but also induces altered cell behaviour. Specifically, extracellular superoxide decreases the magnitude of RBC aggregation, and increases the shear rate required to disaggregate already-formed RBC rouleaux [65]. Given the striking effect that free radicals induce on the deformability and aggregation of RBC, it is possible that blood from haemochromatosis patients may also be more susceptible to mechanical stress, based on the potential for high levels of iron-mediated free radical production as discussed earlier. While ongoing studies are investigating this hypothesis, further evaluation of blood products sourced from haemochromatosis donors is required, particularly if the use involves exposure to high shear environments, such as mechanical circulatory support and/or transfusion.

10 Conclusion

Considering that significant impairments in endothelial function and haemorheological characteristics are present in haemochromatosis patients, it is plausible that many of the secondary complications associated with the disease (i.e., diabetes and cardiomyopathy) are precipitated by impairments in oxygen and nutrient delivery. The potential for venesection therapy to reverse the altered blood rheology in haemochromatosis provides a valuable avenue for further investigations. Given the frequency and volume of blood collected during venesection therapy, and if the rheological properties of blood are normalised following initial treatments, this blood may serve as a useful resource for blood product development. Caution is currently required, however, to ensure that blood cells from hemochromatosis patients are rheologically normal and are able to withstand the high shear environments typical of many blood product destinations (e.g., rotary blood pumps and other devices for mechanical circulatory support).

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