The organs mainly involved in heme synthesis are the liver and the bone marrow, although every cell requires heme to function properly. Heme is seen as an intermediate molecule in catabolism of haemoglobin in the process of bilirubin metabolism.
No Tags! Heme biosynthesis Homo sapiens From WikiPathways. In catalases and peroxidases, heme functions in hydrogen peroxide inactivation or activation, respectively, and in tryptophan pyrrolase, it catalyzes the oxidation of tryptophan Kumar and Bandyopadhyay, Furthermore, heme is indispensable for a wide array of other important enzyme systems, such as cyclooxygenase and nitric-oxide synthase Seed and Willoughby, Other than acting as a prostetic group in hemoproteins, heme itself may influence the expression of many genes.
In non erythroid cells, heme regulates its own production by down-regulating heme biosynthesis at the level of the rate-limiting enzyme 5-aminolevulinic acid ALA synthase 1 ALAS1 and by up-regulating its metabolism Yamamoto et al. Conversely, in erythroid cells, heme acts as a positive feedback regulator for its synthesis and inhibits its degradation Sassa, ; Rutherford and Harrison, Heme may control gene expression at almost all levels by regulating transcription, mRNA stability, splicing, protein synthesis, and post-translational modification Ponka, ; Zhu et al.
Genes coding for globins, heme biosynthetic enzymes, heme-oxygenase HO -1, ferroportin, cytochromes, myeloperoxidase, and transferrin receptor are all regulated by heme. Most of these genes are regulated via heme response elements HREs and the mammalian transcription repressor, Bach1 Ogawa et al. Heme responsive elements HREs are located in enhancer regions of genes induced by heme itself. The transcription factor Bach1 interacts with proteins of the Maf-related family and the resulting heterodimer binds HRE elements, thus repressing gene transcription.
Under conditions of intracellular heme accumulation, heme binds to Bach1, thus leading to a conformational change, a decrease in DNA binding activity and finally its removal from HREs Marro et al. In addition, heme regulates differentiation and proliferation of various cell types. It stimulates neuronal differentiation of mouse neuroblastoma cells Ishii and Maniatis, , erythroid differentiation of erythroleukemia cells Granick and Sassa, , formation of erythroid colonies in mouse as well as in human bone marrow cultures Partanen et al.
In contrast to the positive functions of heme, free heme excess can cause cell damage and tissue injury since heme catalyzes the formation of reactive oxygen species ROS , resulting in oxidative stress. Heme that is not bound to proteins is considered the labile heme pool; this portion of heme is derived from newly synthesized heme that has not yet been incorporated into hemoproteins, or heme that has been released from hemoproteins under oxidative conditions.
ROS damage lipid membranes, proteins and nucleic acids, activate cell signaling pathways and oxidant-sensitive, proinflammatory transcription factors, alter protein expression, and perturb membrane channels Vercellotti et al. Heme toxicity is further exacerbated by its ability to intercalate into lipid membranes.
Due to its lipophilic nature, heme may initially lodge within the hydrophobic phospholipid bilayer. Within this highly oxidizable matrix, iron catalyzes the oxidation of cell membrane and promotes the formation of cytotoxic lipid peroxide, which enhances membrane permeability, thus promoting cell lysis and death Balla et al. Additionally, heme is a potent hemolytic agent.
It affects erythrocyte membrane stability as a result of ROS formation and oxidative membrane damage. Finally, heme is strongly pro-inflammatory since it induces the recruitment of leukocytes, platelets and red blood cells to the vascular endothelium, it oxidizes low-density lipoproteins and it consumes nitric oxide, thus impairing vascular function Figure 1. Figure 1. Free heme toxicity. Free heme has potentially toxic properties due to the catalytic active iron atom it coordinates.
Here, toxic effects of heme are depicted. Additionally, heme is a source of iron. Therefore, heme overload leads to intracellular accumulation of iron, with further ROS generation. Heme is a hemolytic agent 2 , since it intercalates red blood cell membrane, thus favoring cell rupture and further amplifying the hemolytic process. Heme promotes inflammation 3 , by stimulating inflammatory cell activation and cytokine production. Finally, heme causes endothelial dysfunction 4 by several mechanisms: increasing adhesion molecule expression and endothelial activation, promoting inflammatory cell recruitment and platelet aggregation, causing nitric oxide NO oxidative consumption and vasoconstriction, oxidizing low-density lipoprotein LDL.
When intracellular heme accumulation occurs, heme is able to exert its pro-oxidant and cytotoxic action. The cellular free heme pool may increase after extracellular heme overload, increased heme synthesis, accelerated hemoprotein breakdown, impaired incorporation into apo-hemoproteins, or impaired HO activity, resulting in ROS formation, oxidative damage and cell injury.
Several pathological conditions are associated with hemolysis or myolysis, and tissues can subsequently be exposed to large amounts of free heme sickle cell anemia, thalassemia, malaria, paroxysomal nocturnal hemoglobinuria, etc. Gozzelino et al. In summary, heme is a double-faced molecule: physiological amounts of heme act in gene regulation or as the functional group of hemoproteins, providing essential cellular functions, whereas excessive free heme levels result in oxidative stress and tissue injury.
Therefore, the amount of free heme must be tightly controlled to maintain cellular homeostasis and avoid pathological conditions. To this purpose, mammals evolved several defense mechanisms to specifically counteract free heme-mediated oxidative stress and inflammation Wagener et al.
Here the mechanisms involved in the maintenance of heme homeostasis and in the control of heme levels are reviewed: the regulation of both extracellular and intracellular heme content is described, with particular emphasis on the emerging role of heme transporters. Mammals are equipped with various systems able to prevent extracellular heme toxicity. Among them, a key function is covered by the soluble scavengers of free hemoglobin and heme, Haptoglobin Hp Schaer and Buehler, ; Schaer et al.
During several pathological conditions, red blood cells undergo hemolysis and hemoglobin and heme are released into the circulation. Once released, free hemoglobin is captured by its carrier Hp and transported to the macrophages of the reticulo-endothelial system, where the complex is bound by the scavenger receptor CD When the buffering capacity of plasma Hp is exceeded, hemoglobin is quickly oxidized to methemoglobin, which releases free heme Ascenzi et al.
Ferriheme is then bound by Hx, by virtue of its high affinity Figure 2. Hx is a kDa acute phase plasma glycoprotein able to bind an equimolar amount of heme and to transport it into the circulation.
Hx is expressed mainly in the liver, but also in the brain and retina Tolosano et al. Hx is an acute phase response protein. The acute phase response is a complex systemic early-defence system activated by trauma, infection, stress, neoplasia, and inflammation. Most of these stimuli, in particular hemolytic stress and inflammatory stimuli, induce Hx synthesis Tolosano and Altruda, Hx functions as a heme scavenger, maintaining lipophilic heme in a soluble state in aqueous environment and is essential in the re-utilization of heme-bound iron and prevention of heme-induced oxidative damage and cell death Eskew et al.
Hx has the specific function to deliver heme to hepatocytes where the heme-Hx complex is internalized by receptor-mediated endocytosis. To date, the only known Hx receptor is the LDL receptor-related protein 1 LRP 1 , a multi-ligand scavenger receptor present on the surface of many cell types.
Some studies have suggested that Hx can be recycled as an intact molecule to the extracellular milieu. However, Hvidberg et al. The binding of Hx to free heme limits the amounts of heme available as a catalyst of radical formation, makes the essential iron unavailable to invasive microorganisms and contributes to the recycling of iron, as heme iron enters the intracellular iron pool.
Figure 2. Control steps in heme metabolism. The main mechanisms involved in the control of heme levels outside, inside and across the cell are illustrated. After synthesis, heme is exported out of the mitochondrion to the cytosol by the mitochondrial heme exporter FLVCR1b. Thus, the protective role of albumin against heme toxicity remains uncertain Tolosano et al.
The control of intracellular heme content occurs at multiple levels. Here we focus on the regulation of heme synthesis, degradation and plasma membrane heme trafficking, which ensure the maintenance of appropriate intracellular heme concentration.
Animal models discussed in this section are reported in Table 1. Table 1. Mouse models deficient for genes involved in the control of heme homeostasis. The first level of regulation of cellular heme content occurs at the level of heme synthesis control. Heme is synthesized through a series of eight enzymatic reactions Figure 2.
Work in the past decade has shown that heme synthesis is an almost ubiquitous process. There are two isoforms of ALAS: Alas1 gene is located on chromosome 3 in humans and codes for an ubiquitously expressed protein whereas Alas2 gene is on the X chromosome and codes for an erythroid-specific protein Bishop et al. The two isoforms of ALAS mainly differ for their mode of regulation, as discussed later in this section.
ALA is exported in the cytosol soon after its synthesis. The precise molecular mechanism by which ALA is transported through the two mitochondrial membranes is not completely understood. Thus, it has been suggested that SLC25A38 could facilitate the production of ALA by importing glycine into mitochondria or by exchanging glycine for ALA across the mitochondrial inner membrane.
Recently, Bayeva et al. Both proteins are located on the inner mitochondrial membrane; the ALA transporter on the outer mitochondrial membrane still remains to be identified. In the cytosol, two molecules of ALA are condensed to form the monopyrrole porphobilinogen, a reaction catalyzed by aminolevulinate dehydratase ALAD. Then, the enzyme hydroxymethylbilane synthase HMBS catalyzes the head-to-tail synthesis of four porphobilinogen molecules to form the linear tetrapyrrole hydroxymethylbilane which is converted to uroporphyrinogen III by uroporphyrinogen synthase UROS.
All the remaining steps of heme biosynthesis take place inside mitochondria, thus CPgenIII needs to be transported in the mitochondrial intermembrane space. However, data concerning the localization and function of ABCB6 in mitochondria are controversial. ABCB6 was also found to be expressed on the plasma membrane, in the Golgi compartment and in lysosomes. Once synthesized, heme must be exported through the two mitochondrial membranes for incorporation into hemoproteins.
The only mitochondrial heme exporter identified to date is the mitochondrial isoform of Flvcr1 Feline Leukemia Virus subgroup C Receptor 1 gene. FLVCR1b derives from an alternative transcription start site located in the first intron of the Flvcr1 gene thus resulting in the production of a shorter protein Chiabrando et al.
Flvcr1a transcript codes for a protein with 12 transmembrane domains Tailor et al. The role of FLVCR1b as a mitochondrial heme exporter is suggested by in vitro data indicating that its overexpression promotes heme synthesis whereas its silencing causes detrimental heme accumulation in mitochondria. According to its role as a mitochondrial heme exporter, FLVCR1b is essential for erythroid differentiation both in vitro and in vivo as discussed in section Control of Heme Export.
The submitochondrial localization of FLVCR1b is still unknown as well as its ability to interact with other mitochondrial transporters.
Further work is needed to definitively understand how heme is transported across the two mitochondrial membranes. The importance of controlling the rate of heme synthesis is highlighted by the fact that mutations in many genes coding for enzymes involved in this pathway cause specific pathological conditions characterized by the accumulation of toxic heme precursors as discussed in section Pathological Conditions Associated with Alterations of Heme Synthesis.
The regulation of heme synthesis mainly occurs at the level of ALAS, the first and rate-limiting enzyme of the heme biosynthetic pathway. It has been demonstrated that heme plays an essential role in the regulation of its own synthesis by regulating the expression of ALAS. In non-erythroid cells, heme synthesis is dependent on the activity of ALAS1. It has been reported that ALAS1 is directly regulated by heme levels through several mechanisms: heme negatively controls the transcription Yamamoto et al.
The regulation of ALAS1 by heme represents a crucial negative feedback mechanism to maintain appropriate intracellular heme levels in non-erythroid cells, thus avoiding heme-induced oxidative damage. In erythroid cells, heme synthesis is exclusively dependent on the activity of ALAS2. Contrary to ALAS1, heme does not inhibit the expression of ALAS2, as high amount of heme are required for the differentiation of erythroid progenitors. The expression of ALAS2 is controlled at multiple levels.
At the post-transcriptional level, the expression of ALAS2 is regulated by iron availability. This mechanism ensures the coordination of heme synthesis to the availability of iron thus avoiding the production of potentially toxic heme precursors when iron concentrations are limiting. This latter process is dependent on iron but also on heme availability. Thus, during the differentiation of erythroid progenitors, increased cellular iron level stimulates the translation of Alas2 mRNA by inducing the degradation of IRPs.
The following accumulation of heme contributes to the oxidation and degradation of IRP2, further enhancing heme synthesis. This positive feedback mechanism allows a sustained production of heme for hemoglobin synthesis in differentiating erythroid progenitors. The rate of de novo heme synthesis has to be proportionate to its rate of incorporation into newly synthesized apo-hemoproteins. This is obtained at different levels via the control of heme synthesis as well as apo-hemoproteins synthesis.
This evolutionary conserved strategy prevents intracellular heme accumulation, presumably limiting heme cytotoxicity Figure 2. HO is the primary enzyme involved in heme degradation and plays an important role in the protection of cells from heme-induced oxidative stress. Biliverdin is then reduced to bilirubin by the enzyme biliverdin reductase. HO-1 is highly inducible by a variety of stimuli including oxidative stress, heat shock, hypoxia, ischemia-reperfusion, lipopolysaccharide, heavy metals, cytokines and its substrate heme.
Heme is the most potent physiologic inducer of HO The enzyme activity was shown to increase in many tissues, including the liver, kidney, adrenals, ovaries, lung, skin, intestine, heart, and peritoneal macrophages.
HO-2 is ubiquitously expressed and participates in the normal heme capturing and metabolism. The isoenzymes HO-1 and HO-2 are products of two different genes. HO-3 has poor heme-degrading capacity Wagener et al. HO-1 plays a vital function in heme degradation and protects against heme-mediated oxidative injury. Overexpression of HO-1 is associated to the resolution of inflammation through the generation of beneficial molecules like CO, bilirubin, and ferritin resulting from catabolism of toxic heme Wagener et al.
Bilirubin efficiently scavenges peroxyl radicals, thereby inhibiting lipid peroxidation, attenuating heme-induced oxidative stress, cell activation and death Dore et al. CO controls the activity of several heme proteins and causes vasodilation. It also exerts anti-inflammatory effects by inhibiting the expression of pro-inflammatory cytokines Ndisang et al. Finally, ferritin, by sequestering toxic free iron, limits microrganism growth and ROS production.
HO-1 confers cytoprotection against different forms of programmed cell death, including apoptosis driven by heme and tumor necrosis factor Gozzelino and Soares, CO can exert cytoprotective effects via the modulation of cellular signal pathways, including the p38 mitogen activating protein kinase Ndisang et al.
In addition, CO can bind Fe in the heme pockets of hemoproteins, inhibiting heme release and preventing its cytotoxic effects Seixas et al. On the other hand, biliverdin has been described to participate in an antioxidant redox cycle in which, once produced by HO, biliverdin is reduced to bilirubin by biliverdin reductase.
This is followed by the subsequent oxidation of bilirubin by ROS back to biliverdin, forming a catalytic antioxidant cycle that is driven by NADPH, the reducing cofactor of biliverdin reductase. This cycle has the ability to strongly suppress the oxidizing and toxic potential of hydrogen peroxide and other ROS, thus acting as one of the most powerful anti-oxidant physiological system.
Another detoxifying system is represented by ferritin, an evolutionarily conserved Fe sequestering protein that acts as the major intracellular depot of non-metabolic iron Balla et al. Ferritin is a multimeric protein composed of 24 subunits of two types, the heavy chain H-Ft and the light chain L-Ft and has a very high capacity for storing iron up to mol of iron per mol of ferritin.
H-Ft manifests ferroxidase activity that catalyses the oxidation of ferrous iron to ferric iron, thus favoring its storage in L-Ft Hentze et al. Together, HO and ferritin allow rapid shifting of iron from heme into ferritin core where it is less available to catalyze deleterious reactions.
By increasing the expression of HO-1 and ferritin, cells can survive to lethal heme-induced oxidative stress Balla et al. Recent evidence demonstrated that also heme export out of the cell significantly contributes to the regulation of intracellular heme levels. FLVCR1a was originally identified and cloned as a cell-surface protein receptor for feline leukemia virus subgroup C, causing pure red blood cell aplasia in cats Tailor et al.
It was initially reported that FLVCR1a plays an essential role during erythropoiesis, by preventing the toxic accumulation of heme in erythroblasts Keel et al. This hypothesis was suggested by the observation that mice lacking Flvcr1 die in utero due to an impairment of erythroid differentiation at the proerythroblast stage. Similarly, post-natal mice lacking Flvcr1 show a block of erythroid maturation leading to hyperchromic, macrocytic anemia and reticulocytopenia Keel et al.
The specific expression of the two FLVCR1 isoforms in these mouse models still remain to be experimentally verified. Taken together, these data suggest that FLVCR1b, by exporting heme from mitochondria, is essential for fetal erythroid differentiation Chiabrando et al.
Figure 3. The loss of the heme exporter FLVCR1a in mice causes embryonic lethality, skeletal malformation and extended hemorrhages. At this stage, the wild-type embryo shows normal skeletal structure, with fully formed limbs. Figure 4. A Erythroid progenitors are able to synthesize and handle high amount of heme, in view of their hemoglobin Hb -mediated oxygen transport activity. FLVCR1b acts as a mitochondrial heme exporter to allow newly formed heme release from the mitochondrion to the cytosol, where it is incorporated into hemoproteins.
FLVCR1a has been described as a system involved in the control of heme levels inside erythroid progenitors. By mediating heme export out of these cells, FLVCR1a regulates intracellular heme amount, thus limiting free heme toxicity and oxidative damage. B Hepatocytes have the highest rate of heme synthesis after the erythroid progenitors. Hepatic heme is mostly used for synthesis of P enzymes, which metabolize endogenous compounds and xenobiotics. FLVCR1a mediates heme export out of hepatocytes, thus maintaining hepatic heme homeostasis and controlling cell oxidative status.
FLVCR1a export function allows the maintenance of a proper cytosolic heme pool that matches cell need for new hemoprotein generation e. Block of heme export causes heme pool expansion leading to the inhibition of heme synthesis and the reduction of cytochrome activity. Endothelial cells are highly sensitive to heme overload and, in this context, FLVCR1a function could be of crucial importance to export heme excess, thus maintaining heme homeostasis and controlling heme-induced oxidative stress.
As FLVCR1a is ubiquitously expressed, it has been hypothesized that its heme export activity could be relevant in different tissues. Post-natal mice lacking Flvcr1 show iron overload in hepatocytes, duodenal enterocytes and splenic macrophages Keel et al.
Additionally, they show HO and ferritin upregulation, together with alteration of the hepatic oxidative status and induction of the antioxidant genes. These data suggest that heme is normally exported intact from these cells and that in the absence of FLVCR1a, the heme degrading and iron storage systems are upregulated as an attempt to compensate for the lack of heme export Figure 4B.
Interestingly, FLVCR1a expression was found upregulated during cytochrome induction, suggesting that hepatic heme export activity of FLVCR1a was closely associated with heme biosynthesis required to sustain new cytochrome synthesis. Indeed, the lack of FLVCR1a in hepatocytes caused the expansion of the cytosolic heme pool that was responsible for the early inhibition of heme synthesis and increased degradation of heme.
As a result, the expression as well as the activity of cytochromes P was reduced. These findings indicate that FLVCR1a-mediated heme export is crucial to control intracellular heme levels that in turn regulate heme synthesis, thus determining cytochrome function in the liver Vinchi et al. This is particularly evident in the limbs and tail, where vessels do not form properly and branching is severely compromised Chiabrando et al. The molecular mechanism leading to the observed phenotype is still unknown.
Interestingly, FLVCR1a is regulated at the transcriptional level by hypoxia, which has a well-established role in angiogenesis and vasculogenesis Fiorito et al.
FLVCR1a mediated heme export could work in strong association with HO-1 to determine the appropriate amount of heme in endothelial cells. It is well established that HO-1 plays a pivotal role in the regulation of vascular biology Belcher et al.
For this reason, it will be interesting to investigate the role of FLVCR1a in hemolytic disorders characterized by enhanced heme-induced oxidative stress in endothelial cells. To date, it has been reported that FLVCR1a localizes on the sinusoidal membrane of hepatocytes, likely exporting heme in the bloodstream Vinchi et al. These results have been obtained by studies on the overexpressed protein and are limited by the lack of specific antibodies for this exporter that prevents any analysis of the localization of endogenous FLVCR1a.
Future work is required to specifically address FLVCR1a localization in different cell types in vivo. The Abcg2 gene is located on human chromosome 4q22 and it consists of 16 exons and 15 introns Bailey-Dell et al.
Only the homodimer is functional Kage et al. ABCG2 is localized at the plasma membrane and it is expressed in several tissues including hepatic canalicular membranes, renal proximal tubules, intestinal epithelium and placenta Doyle and Ross, Moreover, ABCG2 is expressed in a sub-population of hematopoietic stem cells.
It prevents cytotoxicity of chemotherapics and confers resistance to hypoxic conditions Krishnamurthy et al. Recently, genetic studies in human disease established that ABCG2 functions as a urate transporter that promotes urate excretion in the kidney as discussed in section Hyperuricaemia and Gout Qiu et al.
This was caused by the accumulation of pheophorbide, a degradation product of chlorophyll present in the diet, structurally similar to PPIX. Since Abcg2 expression is induced by HIF1, ABCG2 is thought to confer a strong survival advantage to stem cells under hypoxic stress by reducing intracellular porphyrin content Krishnamurthy et al. Alternatively, this could also indicate that ABCG2 is not a physiologic porphyrin exporter.
Based on its localization at the apical membrane of duodenal enterocytes, it has been hypothesized that ABCG2 could transport excess heme or porphyrin from the enterocyte to the lumen. If, other than in urate transport and in drug metabolism, ABCG2 plays a role in heme export under physiologic or pathologic conditions remains to be elucidated.
A further level of control of intracellular heme content is represented by the modulation of heme import inside the cells Figure 2. To date the only known proteins with a well-established function as heme importers are HRGs Rajagopal et al. The SLC49 family belongs to the Major Falicitator Superfamily MFS of secondary active permeases that transports small solutes across membranes in response to chemico-osmotic gradients, contributing to the maintenance of normal cell homeostasis Pao et al.
A variant transcript exists, encoding a shorter hypothetical protein that differs in the N-terminal and whose significance is unknown Brown et al. Flvcr2 mRNA is found ubiquitously, with the highest transcript levels observed in human brain, placenta, lung, liver, kidney and hematopoietic tissues Duffy et al. In mouse, its expression has been reported in brain, spinal cord Lein et al.
The FLVCR2 protein is composed by 12 predicted transmembrane domains and six presumptive extracellular loops, it is not N-linked glycosylated and shows a molecular mass of about kDa, while its truncated variant weights about 40 kDa Brown et al. Unfortunately, to date a FLVCR2-specific antibody is not available, so protein expression in the different tissues and cell compartments has not been determined.
Nevertheless, the FLVCR2 functions, described below, strongly indicate its localization on the cell plasma membrane.
Brasier et al. Although this function has not been formally excluded by subsequent studies, alternative roles for FLVCR2 have emerged. Finally, genetic studies in humans associated Flvcr2 mutation to the Fowler syndrome, a disorder with no obvious link to heme metabolism see section Fowler Syndrome. Thus, the assumption that FLVCR2 is a heme importer is not definitive and further studies are needed to fully address the substrate specificity of this transporter.
No expression was observed in mouse placenta and ileum Shayeghi et al. Both transcripts were detected in kidney, liver, placenta, duodenum and spleen, and to a lesser extent in jejunum, ileum, cecum, colon, rectum, and testis. In non-polarized cells, it could also localize in the lysosome Yanatori et al. Nevertheless, Qiu et al. Considering that the K m at pH 6. Moreover, it has been proposed that it could be involved in the export of hemoglobin-derived heme from the endosome to the cytoplasm of macrophages Schaer et al.
Nevertheless, the prevalent hypothesis is that, as for folates uptake, it could utilize the co-transport of protons along a favorable concentration gradient to drive the transport of heme Shayeghi et al. Many pathological conditions are associated with hemolysis and extracellular heme release. In all these conditions, the pool of circulating Hx is diminished and, consequently, the plasma heme scavenging capacity is strongly reduced.
In the last decade, the use of a knockout mouse model for Hx was valuable to elucidate its function as well as its potential use as a therapeutic molecule in the prevention of heme adverse effects Table 1. Additionally, other functions of Hx not clearly related to its role as an heme scavenger, have also been described thanks to the use of knock-out mice Fagoonee et al. Endothelial activation is a proinflammatory and procoagulant state of the endothelial cells lining the lumen of blood vessels.
This state is mainly characterized by an increased expression of adhesion molecules on endothelial cell surface, which promotes the adherence of leukocytes as well as platelets and red blood cells, thus favoring inflammation, clot and eventually thrombus formation.
According to its function as a free heme scavenger, Hx is expected to counteract heme toxicity on the vascular endothelium and increasing experimental evidence is supporting this concept.
This recent observation further strengthens the concept that heme triggers vascular inflammation and damage, and emphasizes the importance of Hx in counteracting heme-driven cardiovascular dysfunction associated with hemolytic conditions. This could have relevance, in the future, for therapeutic interventions against cardiovascular and endothelial dysfunctions in hemolytic patients Vinchi and Tolosano, Malaria is another well-known hemolytic condition, associated with the accumulation of high concentrations of free heme in plasma.
The appearance of hemoglobin and heme in plasma has been linked to the development of cerebral malaria, which remains the most severe and difficult to treat complication of the infection Ferreira et al.
The potential protective effect of both Hp and Hx in this pathology still needs to be elucidated. Recently, hemolysis has been observed to occur after red blood cell transfusion, one of the most common therapeutic interventions in medicine.
Over the last two decades, however, transfusion practices have been restricted, limiting unnecessary transfusions Barr and Bailie, The adverse effects of transfusions seem to be mainly related to the storage period between blood donation and transfusion Wang et al. Transfusions of old blood in animal models result in both intravascular and extravascular hemolysis and cause hypertension, acute renal failure, hemoglobinuria, and vascular injury. Conversely, old blood transfusion together with the hemoglobin scavenger Hp attenuated most of the transfusion-related adverse effects Baek et al.
Whether a similar protection, alone or in association with Hp, could be afforded by Hx still needs to be explored. Not long ago, severe sepsis was found accompanied by hemolysis and Hx exhaustion in mice Larsen et al. Larsen and coworkers showed that the administration of exogenous Hx was protective against organ injury and prevents the lethal outcome of severe sepsis in mice Larsen et al. The protective effect in this model is related to the ability of Hx to counteract heme proinflammatory effects upon pathogen infection.
Furthermore, a neuroprotective effect for Hx was also described. Hx was found expressed by cortical neurons and present in mouse cerebellum, cortex, hippocampus, and striatum.
Upon experimental ischemia, neurologic deficits as well as infarct volumes in the brain were increased in Hx deficient mice, indicating that Hx regulates extracellular free heme levels and the heme-Hx complexes protect primary neurons against the heme-induced toxicity Li et al.
The rationale for the use of Hx as a therapeutic is based on the idea that it acts by scavenging circulating free heme, the ultimate mediator of hemoglobin toxicity. Therefore, replenishing the circulating stores of heme scavengers, thereby compensating for the loss of heme scavenging capacity in plasma, may be used as a therapeutic approach to target circulating free heme and prevent its deleterious effects Schaer and Buehler, ; Schaer et al.
To date, we are still far from the use of heme scavengers as therapeutics and no clinical trials are ongoing. Anyway, the possible use of these molecules for therapeutic purposes has elicited the interest of the research community in the field as well as of several pharmaceutical companies. Pre-clinical studies are ongoing with the aim of translating the protective effects of heme scavengers into clinical practice in the near future.
Eight distinct types of inherited porphyrias have been described, each resulting from a partial deficiency of a specific enzyme of the heme biosynthetic pathway Figure 2 and Table 2. The porphyrias are characterized by an impairment of heme synthesis, leading to the accumulation of specific intermediates of the heme biosynthetic pathway in various tissues. Table 2. Disorders associated to mutations in genes involved in heme metabolism. The initial accumulation of the porphyrin precursors occurs primarily in bone marrow erythroid cells.
Hypochromic, microcytic anemia is also common and the disorders may evolve to severe hepatobiliary disease and hepatic failure Anstey and Hift, ; Whatley et al. Mutations in the UROS gene cause the third form of erythropoietic protoporphyria, called congenital erythropoietic protoporphyria CEP; OMIM: ; the partial loss of UROS activity leads to the incomplete metabolism of hydroxymethylbilane and the accumulation of non-physiologic porphyrin isomers in the bone marrow, erythrocytes, urine and other organs.
CEP is mainly characterized by cutaneous photosensitivity and hemolytic anemia Sassa and Kappas, ; Murphy, ; Bishop et al. Five different types of hepatic protoporphyria have been described.
The major manifestations of these disorders are the life-threatening acute neurologic attacks and abdominal pain Balwani and Desnick, Mutations in the Urod gene cause a cutaneous form of hepatic porphyria, called porphyria cutaneous tarda PCT; OMIM: ; impaired UROD activity leads to the accumulation of uroporphyrin and other highly carboxylated porphyrins in the skin, liver and erythrocytes. PCT is characterized by blistering skin lesions that appear most commonly on the backs of the hands increased cutaneous photosensitivity and liver disease whereas neurologic features are usually absent Frank and Poblete-Gutierrez, ; Balwani and Desnick, The generation of animal models of porphyrias allowed a better understanding of the pathophysiological mechanisms involved in the distinct types of porphyria as well as the development of novel therapeutic strategies Richard et al.
Sideroblastic anemias are genetically and clinically heterogeneous disorders characterized by the pathological accumulation of iron in the mitochondria of erythroid precursors. Sauternes sau is a zebrafish mutant characterized by a delay in erythroid differentiation, abnormal globin gene expression and heme deficiency. The absence of Alas2 in mice causes embryonic lethality due to a severe block of erythroid differentiation.
In contrast to human patients, ring sideroblasts are not present and iron deposition occurs in the cytoplasm instead of mitochondria Nakajima et al. Interestingly, other forms of inherited sideroblastic anemias are due to mutations in genes indirectly involved in the heme biosynthetic pathway. Mutations in the gene coding for SLC25A38 , the putative mitochondrial exporter of ALA, have been identified in patients with an autosomal recessive form of sideroblastic anemia Guernsey et al.
Iron-sulfur clusters deficiency causes the activation of IRP1, mitochondria iron accumulation and cytosolic iron depletion that in turn activates IRP2. Thus, a primary defect in iron-sulfur clusters biogenesis secondarily affects heme synthesis in erythroblasts, resulting in mitochondrial iron loading and the same pathophysiology of ALAS2 deficiency. These mice were characterized by serum iron deficiency and pathologic tissue iron-loading, indicating that HO-1 is crucial for the expulsion of iron from tissue stores and for its reutilization.
These data demonstrated that, although HO-1 is a stress-induced protein, it is important under basal conditions to protect liver and kidney from oxidative damage and that it is an essential regulator of iron metabolism and homeostasis. Additionally, these mice suffered from delayed growth and progressive chronic inflammatory diseases as suggested by an enlarged spleen and lymph nodes, hepatic inflammatory cell infiltrates, vasculitis, and glomerulonephritis.
Furthermore, they were found to be extremely sensitive to oxidative injury and prone to hepatic necrosis and death upon lipopolysaccharide administration. In the spleen, initial splenic enlargement was observed to progress to red pulp fibrosis, atrophy, and functional hyposplenism in older mice. Finally, the failure of tissue macrophages to remove senescent red blood cells led to intravascular hemolysis, circulating hemoglobin release, and iron redistribution to hepatocytes and kidney proximal tubules.
Indeed, the lack of HO-1 strongly impairs macrophage function, thus causing iron redistribution and severe oxidative tissue injury. The sequence analysis of the HO-1 gene revealed the complete loss of exon 2 on the maternal allele and a 2-nucleotide deletion in exon 3 on the paternal allele. The disease was reported in a 6-year-old boy, who suffered from severe growth retardation, asplenia, marked hepatomegaly, renal injury, tissue iron deposition and paradoxically elevated Hp levels. Moreover, he showed increased red blood cells fragility, chronic hemolysis, anemia, leukocytosis, thrombocytosis, disseminated intravascular coagulation, hyperlipidemia and mesangio-proliferative glomerular changes, likely resulting from endothelial injury and reticulo-endothelial dysfunction.
The serum level of Hp is usually reduced in hemolytic states; in this patient suffering from hemolytic anemia, however, the Hp level was rather increased.
In the HO-1 deficient case, Hp production rather than its consumption could be increased due to a dominant effect of inflammation. In addition, reticuloendothelial dysfunction could delay the clearance of the Hp-hemoglobin complex. Histologically, the fibrous plaques were characterized by the proliferation of smooth muscle cells and few foam macrophages. The enhanced proliferation of smooth muscle cells could be a consequence of HO-1 loss.
Recently, another case of HO-1 deficiency was described in a year-old girl who presented massive hemolysis, inflammation, nephritis and congenital asplenia Radhakrishnan et al. The key features that suggested HO-1 deficiency were marked hemolysis, generalized inflammation with evidence of endothelial injury and nephropathy with underlying asplenia. Mutation analysis showed the presence of homozygous missense mutations in exon 2 R44X on chromosome 22q12, which resulted in the absence of the functional HO HO-1 deficiency in humans is characterized by total asplenia and this is fully recapitulated by the mouse model of HO-1 deficiency suggesting that this enzyme has a key role in macrophage heme metabolism and heme-iron reutilization.
On the other hand, compared with the knockout mouse model, the human cases of HO-1 deficiency were observed to involve more severely the endothelial cells. As the name of the disease suggests, it is not always manifest but only intermittently. Heme is the prosthetic group of cytochrome P enzymes, which are important in drug metabolism and are induced in the liver by various drugs see slide It appears that ALA synthase is induced along with the cytochrome P enzymes, and AIP attacks are often triggered or aggravated by the application of such drugs.
Specific drugs that induce cytochrome P and ALA synthase include barbituric acid derivatives and carbamazepine, which were, and occasionally still are, used in the treatment of psychiatric symptoms. Fatal outcomes have occurred when AIP patients were misdiagnosed and treated with barbituric acid derivatives. Red blood cells have a regular lifespan of days although it can be considerably shorter in some diseases.
At the end of this lifespan, they are captured and ingested by phagocytes in the spleen and the liver. When the globin protein is proteolytically degraded, heme is released. Heme itself undergoes degradation mostly in the liver.
Ring cleavage by heme oxygenase produces biliverdin, which is in turn reduced to bilirubin. Some bilirubin is excreted into the bile as such; however, the greater share is first conjugated with glucuronic acid by UDP-glucuronosyltransferase, form 1A1, and excreted thereafter. The major transport protein responsible for excretion of the diglucuronide is an ABC transporter ABCC2 , the same one that also secretes bile acids see slide In the anaerobic environment that prevails inside the colon, the released bilirubin subsequently undergoes reduction, again by bacterial enzymes, to variously colored pigments that produce the stool color.
Another reduction product, urobilinogen, is taken up and excreted with the urine, causing the yellow color of the latter. Some fairly simple clues can narrow down the cause of jaundice in a given patient. If excretion of bilirubin is blocked, the pigments derived from it will be absent, and the stool will have a grayish color. Hemolysis consists in the accelerated decay of red blood cells; it may result from biochemical causes such as glucosephosphate dehydrogenase deficiency see section 9.
In hemolysis, the serum level of unconjugated bilirubin will be more strongly increased than that of the diglucuronide. On the other hand, when the flow of the bile is backed up, the conjugated bilirubin will spill back into the serum and will be increased. Liver diseases can affect synthesis, conjugation and biliary secretion of bilirubin in various degrees, and either form of bilirubin can be more strongly increased than the other.
Neonatal jaundice is a normal event that is caused by a transiently low level of UDP-glucuronosyltransferase 1A1. If the serum level of bilirubin gets too high, however, it may accumulate in the brain and cause neurological problems see next slide. To prevent this, newborns can be treated with phototherapy see slide As in many other gene defects, there are variants with total or partial disruption of enzyme activity. When residual enzyme activity is present, it is possible to increase it with drugs such as phenobarbital that transcriptionally induce it.
As in neonatal jaundice, phototherapy is also used in Crigler-Najjar syndrome, but its efficiency decreases with time, since the growth of the body reduces its surface to volume ratio, and therefore a diminishing fraction of the bilirubin in the body can be reached by illumination.
The disease is best treated with liver transplants, as the transplanted liver will not be affected by the underlying gene defect and be able to conjugate and excrete bilirubin. This slide shows a brain section from a patient with severe bilirubin encephalopathy. The yellow color in the deeper structures of the forebrain, the so-called basal ganglia , is due to bilirubin accumulation.
Through an unknown biochemical mechanism, bilirubin causes damage to the basal ganglia, which results in motor dysfunction and other neurological symptoms. In phototherapy, bilirubin absorbs photons and subsequently undergoes cis-trans isomerization across the two remaining double bonds between the pyrrole rings of the bilirubin molecule, as well as ring formation [ ]. This slide shows some of the photochemical reaction products. The 4Z,15Z isomer of bilirubin top left is the one that is produced directly by biliverdin reductase, and which is eliminated very slowly in the unconjugated form.
The other isomers are eliminated more rapidly; the fastest rate of elimination is observed with lumirubin cyclobilirubin. While the absorption maximum of bilirubin is in the blue wavelength band, green light reportedly produces lumirubin more efficiently, and it also induces less cytotoxic byproducts in cell culture models [ ].
It seems, however, that blue lamps are still more widely used in practice, and the literature does not make mention of significant side effects of blue light, even in the long-term treatment of Crigler-Najjar patients [ ].
The inhibition of heme oxygenase with Sn-mesoporphyrin has been used successfully in clinical studies to treat neonatal jaundice. The study summarized in this slide examined the effectiveness of Sn-mesoporphyrin in the treatment of newborns with glucosephosphate dehydrogenase deficiency see slide 9. In this condition, the lifespan of red blood cells is diminished, which increases the rate of heme degradation; newborns therefore are at an increased risk of severe jaundice.
Remarkably, a single injection of the drug was sufficient to reduce the peak levels of bilirubin to a greater extent than the reference treatment phototherapy. Phototherapy currently remains the standard treatment in clinical practice. Figure prepared from original data in [ ].
Nitric oxide is an important signaling molecule. As a small molecule, it can easily diffuse out of one cell and into another. Inside the target cell, it binds to soluble guanylate cyclase sGC. Interestingly, the NO-binding site on sGC is a heme molecule. Binding of NO to one face of the heme releases a histidine side chain on the other, which causes a conformational change and activation of the sGC molecule.
In vitro experiments show that CO can also bind and activate sGC. It has therefore been proposed that heme oxygenase, which produces CO, has a regulatory role in addition to its metabolic one.
However, while this idea has been around for awhile, I have not come across solid evidence that supports a significant signaling role of CO in vivo. The very high affinity of transferrin to iron means that there is practically no free iron in the blood serum.
Bacteria, like human cells, require iron for growth; therefore, keeping free iron very low is an important non-specific immune mechanism.
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