SUSTANCIACIÓN DEL JUICIO DE AMPARO DIRECTO

Evidence that iron may exist in plasma unbound to transferrin and that this form of iron may be toxic was first provided by Hershko et al. (1978). Despite considerable skepticism initially, the evidence for the existence of NTBI has now become persuasive. NTBI was firstly recognised as nonspecific iron present in thalassaemic serum, which was chelatable and subsequently dialysable or filtrable (Hershko et al., 1978). It has been characterized as bleomycin-reactive iron present in the plasma of patients with iron-overload which can generate harmful hydroxyl radicals and promote lipid peroxidation (Gutteridge et al., 1985; Halliwell and Gutteridge, 1986). It has been suggested that NTBI is probably a low MW iron complex loosely bound to plasma proteins such as albumin (Hershko and Peto, 1987). An independent study proposed that such NTBI may be ferritin iron (Pootrakul et al.,

1988). By using the HPLC/NMR technique, it has been postulated that NTBI in plasma of patients with iron-overloaded haemochromatosis is mainly present as iron citrate and also iron-citrate-acetate complexes (Grootveld et al., 1989). Singh et al. (1990) hypothesized that NTBI in thalassaemic semm comprises low MW iron complexes and iron loosely bound to semm proteins. Nevertheless, the precise nature of nonspecific NTBI has not been characterized.

Relevance o f N T B I to the pathophysiology o f iron overload in thalassaemia

In patients with p-thalassaemia major, transfusional iron accumulates at a rate of 0.25-0.4 mg/kg body weight per day (Gordeuk et al., 1987). In patients with thalassaemia intermedia in whom regular transfusions are not administered but in whom substantial ineffective erythropoiesis is ongoing, most of the iron released to the circulation is derived from catabolism of red blood cells at a rate approximately eight-fold greater than that observed in normal subjects (0.7 mg/kg/day). Clearly, significant daily iron accumulation places patients with both phenotypes at high risk of iron-induced toxicity (Hershko and Rachmilewitz, 1979). When the capacity of the plasma transferrin is exceeded, excess cataboUc iron emerges in the form of NTBI. This form of iron is highly toxic, resulting in rapid uptake of iron into tissues and if untreated, such tissue loading results in early death, usually from cardiac complications.

Following the first recognition of the potential importance of NTBI (Hershko et al., 1978) subsequent studies have confirmed its existence (Anuwatanakulchai et al., 1984; Gutteridge et al., 1985; Wagstaff et al., 1985; Singh et al., 1990) and demonstrated that this fraction of iron may generate harmful hydroxyl radicals and promote lipid peroxidation

radicals generated by excess iron (including superoxide dismutase, catalase and glutathione reductase) are absent in plasma.

By eliminating NTBI with optimal chelating therapy, the uptake of toxic iron species can, in principle, be prevented. The difficulty is that, because NTBI reappears within minutes of cessation of DFO infusion (Porter et al., 1996), standard chelation regimens may fail to clear NTBI for periods sufficient to prevent iron-mediated tissue damage. This is supported by the findings of pituitary failure and cardiac dysfunction in patients with apparently optimally previous treatment with DFO. The relationship between tissue iron loading, NTBI, organ damage, and standard regimens of iron-chelating therapy therefore requires further careful scrutiny.

While NTBI appears to be poorly correlated with estimates of body iron burden, including the serum ferritin concentration and transferrin saturation (Anuwatanakulchai et al., 1984; Al-Refaie et al., 1992), the relationship between NTBI and quantifications of body storage iron has not been studied systematically. Hitherto, this has not been possible mainly because of technical difficulties in the lehable measurement of NTBI, especially in serum samples in which iron-chelating agents are present. Methodology development allowed measurement of NTBI (Singh et al., 1990; Porter et al., 1996), and its rate of removal and reappearance, during DFO treatment in vivo (Porter et al., 1996). As will be shown later however, this methodology requires further modification.

The concentrations of NTBI and hpid-soluble antioxidants were investigated in p- thalassaemia major patients (Livrea et al., 1996). The results showed that NTBI was in the range of 4.5-54.8 |Lig/dl and had a positive trend with ferritin. The concentration of lipid- soluble antioxidants in these patients was depleted whereas a mild to severe hepatic damage was shown in 24 of 42 patients. These results suggest that the measurement of peroxidation products may be a simple measure of iron toxicity in thalassaemia, in addition to the conventional indices of iron status.

Chemical nature o f N T B I

As discussed above, the exact chemical nature of NTBI in iron overload has been the subject of considerable debate. While low MW NTBI species have been demonstrated both in the absence (Lau and Sarkar, 1984) and presence (Grootveld et al., 1989) of iron overload, NTBI is certainly present at very high concentrations (10'^ M) following acute

iron poisoning, and in patients with chronic iron overload (10 ^ to 10'^ M). In the latter group, although other low MW species have been described (Lau and Sakar, 1984), iron citrate complexes may predominate (Grootveld et al., 1989) but 10'^ M citrate (the concentration found in plasma; Harris, 1992) binds only low concentrations of iron at physiological pH. At higher concentrations, NTBI is likely to be polynuclear or bound to proteins such as albumin. It has also been suggested, though not demonstrated directly, that plasma ferritin may contribute to this higher MW pool (Pootrakul et al., 1978).

Uptake and kinetics o f N T B I

The kinetics of NTBI uptake has been studied in animal and tissue culture models using a variety of forms of iron prepared in vitro (e.g. iron-citrate, iron-NTA). The physiological relevance of these iron forms to the human situation is unclear. Nevertheless, such studies have shown that low MW ferric and ferrous citrate complexes are cleared rapidly in perfused rat liver by an efficient saturable first pass process with Kna value of 14-22 |iM.

The kinetics of higher MW forms of NTBI are presently unknown. NTBI is taken up rapidly by heart cells at 200 times the rate of iron released from transferrin (Link et al., 1989), a process of uptake which, unlike transferrin-iron uptake which is inhibited at high tissue iron concentrations, may actually be increased by high tissue iron content (Randell et al., 1994). A detailed discussion of NTBI uptake mechanisms into liver, heart and other cells has been given in Section I.2.5.2.

Mechanisms o f toxicity by N T B I

The role of iron in promoting the conversion of superoxide and hydrogen peroxide into highly toxic, free hydroxyl radicals through the Haber-Weiss reaction is well documented (Halliwell and Gutteridge, 1986) and discussed below (Section 1,3.3). Previous evidence (Gutteridge et al., 1985) demonstrated that NTBI ultrafiltrates from haemochromatosis sera were capable of promoting the free-radical formation by xanthine oxidase and acetaldehyde, and stimulating the peroxidation of phospholipid liposomes. In the absence of detoxifying intracellular enzymes such as superoxide dismutase, catalase and glutathione reductase, NTBI may, as it has been shown to do in vitro (Gutteridge et al.,

1985), promote the formation of free hydroxyl radicals and accelerate the peroxidation of membrane lipids. Increased lipid peroxidation, usually regarded as the most significant event in the pathogenesis of cellular damage, may target polyunsaturated fatty acids

resulting in the formation of highly reactive aldehydes such as malonyldialdehyde (MDA) and 4-hydroxynonenal (4-HNE), and leading to the formation of covalent links to proteins, or protein adducts (Houglum et al., 1990). As a result of this lipid peroxidation, increased lysosomal fragility has been observed iron-loaded livers (Seymour and Peters, 1978; Weir et al., 1984). NTBI also increases formation of MDA and conjugated dienes and is associated with increased respiratory excretion of low MW alkanes (Goddard and Sweeney, 1983; Dillard et al., 1984).

1.3.3 Pathology of iron m ediated toxicity

1.3.3.1 G eneration of free radicals by iron

Iron is made unavailable to participate in the generation of harmful free radicals by its binding to hgands such as transferrin. Nevertheless, in iron overload, plasma transferrin becomes saturated and NTBI is detectable (Hershko et al., 1978). Also, increased quantities of low MW iron in the cells (e.g. LIP) are potentially available to participate in free radical generating reactions. Iron is particularly important because it is present at sufficient concentration in tissues and due to the favourable redox potential of the Fe^'*^/Fe^'^ transition (between 4-0.35 and 4-0.5 V) which allows easy redox cycling between Fe^'^/Fe^^. Free radicals are defined as any species capable of independent existence that contains one or more unpaired electrons. One of the commonest reactions in the body is the iron- catalysed reduction of molecular oxygen in water to sequentially form products including superoxide (O2 ), hydrogen peroxide (HjOj) and the hydroxyl radical (HO*). The iron- catalysed reaction between superoxide and hydrogen peroxide was first described by Haber and Weiss in 1934 and involves the sequential reduction and reoxidation of Fe^^.

O2 4- H2O2 --- > O2 4- OH 4- HO* Haber-W eiss reaction

The hydroxyl radical (HO*) has also found great use in studying the structural properties of DNA and DNA-protein complexes. This species can be efficiently generated via the Fenton reaction from Fe(II)-EDTA in the presence of hydrogen peroxide and a reducing agent such as ascorbate. Hydrogen peroxide is relatively stable and non-toxic by itself. Furthermore, it is an important precursor of hydroxyl radicals, requiring the availability of catalytic trace elements such as iron, copper or cobalt.

+ O2 <---> + O2*

2 O2*’ + 2 <--- > H2O2 + O2 Fenton reaction

Fe^+ + H2O2 <---> Fe^+ + OH* + OH

The hydroxyl radical can diffuse and abstract hydrogen atoms from DNA, a process that results in cleavage following subsequent chemical reaction. These and other reagents that facilitate DNA or RNA cleavage via hydrogen atom abstraction and free radical chemistry have been and are being identified. In the presence of iron(II) and oxygen molecules or iron(III) and hydrogen peroxide, bleomycin, an anti-tumour antibiotic isolated from the fungus Streptomyces, binds to DNA and induces single- and double-strand cleavages in the DNA molecule. Upon activation, a ferric peroxide or a high-valent species that abstracts hydrogen atoms from the deoxyribose rings of DNA is formed, and subsequently results in DNA cleavage. The various systems differ in that the hydrogen-abstracting agent may be a metal complex, often bound to the DNA (as in bleomycin), or a freely diffusable species such as a hydroxyl radical.

A number of physiological scavengers of toxic oxygen products exist such as superoxide dismutase which oxidises superoxide to hydrogen peroxide, catalase and glutathione peroxidase which scavenge hydrogen peroxide. Tissue damage will depend on the relative rates of formation and scavenging of harmful free radicals. Hydroxyl radical can damage several biomolecules including proteins, DNA and membrane phospholipids. Due to the weakness of adjacent double bonds, polyunsaturated fatty acids of membrane phospholipids are sensitive to peroxidation to form hpid peroxy radical and subsequently lipid peroxide. Lipid peroxides are stable but their decomposition can be catalysed by transition metals and metal complexes to cyclic-peroxides and -endoperoxides, with fragmentation to aldehydes including MDA.

It has been shown that liposome peroxidation depends on their charge (Kunimoto et al., 1981). The site of peroxidation and whether the iron or hydroxyl radical is membrane associated will affect the incidence of any free radical producing reaction. Possibly, the protein proportion of normal cell membranes (up to 50%) will influence propagation of lipid reactions. Hence, cell systems are likely more suitable for studying iron mediated lipid peroxidation than noncellular systems. EDTA-Fe(II) complexes do not diminish the reactivity of iron salts in the Fenton reaction and may catalyse peroxidation (Gutteridge and

Halliwell, 1989). In comparison DFO tightly binding to wiU completely inhibit lipid peroxidation in several systems (Gutteridge et al., 1979).