Chapter 17 Disorders of haemoproteins, porphyrins and iron
Haemoglobin, the oxygen-carrying pigment of blood, consists of a protein, globin, and four haem molecules. Globin comprises two pairs of polypeptide chains (the principal haemoglobin in adults, haemoglobin A (HbA) has two α- and two β-chains); each polypeptide binds one haem molecule. Haem (Fig. 17.1) consists of a tetrapyrrole ring, protoporphyrin IXα, linked to an iron II ion (Fe2+), to which oxygen becomes reversibly bound during oxygen transport. Other haemoproteins include myoglobin, which binds oxygen in skeletal muscle, and the cytochromes, enzymes responsible for catalysing many oxidative processes in the body.
The major part of the body’s iron is present in haemoglobin and the major product of porphyrin metabolism is haem. It is thus convenient to discuss the chemical pathology of the haemoproteins, the porphyrins and iron together, although disorders affecting any one of these do not necessarily (indeed do not often) involve the others.
Haemoglobin and haemoglobinopathies
Haemoglobin is a very accessible protein and has been studied extensively. The haemoglobinopathies (genetically determined abnormalities of haemoglobin synthesis) fall into two groups: qualitative, most frequently involving amino acid substitutions, and quantitative disorders, known as ‘thalassaemias’.
Amino acid substitutions
Here there is a single amino acid substitution in one of the polypeptide chains. Over 200 such variants have been described. Some of these involve amino acids that are not structurally or functionally vital, and as a result are clinically silent; others have important consequences, including effects on haemoglobin solubility (e.g. HbS, the haemoglobin of sickle cell disease), stability and oxygen-carrying capacity.
In the thalassaemias there is an inherited defect in the rate of synthesis of one of the globin chains. This can involve the α-chains (α-thalassaemia) or the β-chains (β-thalassaemia). The consequences include ineffective erythropoiesis, haemolysis and a variable degree of anaemia. The clinical severity varies between the different thalassaemias. Some are clinically silent except during periods of stress, such as severe infection or pregnancy, when anaemia may develop, while others cause severe, persistent anaemia. When α-chain synthesis is totally absent, affected infants are either stillborn or die shortly after birth.
The investigation and management of the haemoglobinopathies is the province of the haematologist, and these disorders are not considered further in this book.
Abnormal derivatives of haemoglobin
Methaemoglobin is oxidized haemoglobin, with iron in the Fe3+ form. It is incapable of carrying oxygen. A small amount is normally produced spontaneously in red blood cells but can be enzymatically reduced back to haemoglobin. Excessive methaemoglobin (methaemoglobinaemia) can be congenital or acquired. It can occur in some haemoglobinopathies, with an inherited deficiency of the reductase enzyme, and also as a result of the ingestion of large amounts of certain drugs, such as sulphonamides. In toxic methaemoglobinaemia, the presence of methaemalbumin (formed as a result of haemolysis of red cells containing methaemoglobin) imparts a brown colour to the plasma, while the presence of free methaemoglobin may give the urine a similar colour.
The major clinical manifestation of congenital methaemoglobinaemia is cyanosis. Acute toxic methaemoglobinaemia causes symptoms of anaemia and may lead to vascular collapse and death. Methaemoglobinaemia, except when due to a haemoglobinopathy, can be treated with methylthioninium chloride (methylene blue) or ascorbic acid, agents that reduce the abnormal derivative back to haemoglobin.
Sulphaemoglobin, a group of poorly characterized derivatives of haemoglobin, is often formed together with methaemoglobin. It is also incapable of carrying oxygen, but cannot be converted back to haemoglobin.
Carboxyhaemoglobin (COHb) is formed from haemoglobin in the presence of carbon monoxide, the affinity of the pigment for this gas being some 200 times greater than for oxygen. Because of this, only small quantities of carbon monoxide in the inspired air can result in the formation of large amounts of COHb, and hence greatly reduce the oxygen-carrying capacity of the blood. The binding of carbon monoxide to haemoglobin also causes a left shift in the oxyhaemoglobin dissociation curve (see p. 58), decreasing the availability of oxygen to tissues. Small amounts of COHb (<2%) are commonly present in the blood of urban dwellers and greater amounts (up to 10%) may be found in the blood of tobacco smokers.
Measurement of COHb is critical to the diagnosis of carbon monoxide poisoning. Carbon monoxide is produced by the incomplete combustion of fuels. Self-exposure to motor car exhaust in a confined space (e.g. a closed garage) is a well-recognized means of suicide and occasionally homicide. Chronic carbon monoxide poisoning is usually the result of poorly fitted or maintained gas appliances in the home.
Haematin is oxidized (Fe3+) haem. It is released from methaemoglobin when red cells containing this pigment are haemolysed, but can be formed from free haem in severe intravascular haemolysis. Haematin combines with albumin in the bloodstream to form methaemalbumin. Methaemalbuminaemia is sometimes a feature of acute haemorrhagic pancreatitis.
Protoporphyrin IXα, which combines with iron to form haem, is the end product of a series of complex reactions. The first step that is unique to this pathway is the combination of glycine and succinyl CoA to form δ-aminolaevulinic acid (ALA), a reaction catalysed by the enzyme ALA synthase (Fig. 17.2). Two molecules of ALA then condense to form porphobilinogen (PBG), in a reaction catalysed by PBG synthase (also known as ALA dehydratase).
Figure 17.2 The biosynthesis of porphyrins. PBG deaminase is also known as hydroxymethylbilane synthase and ALA dehydratase as PBG synthase.
The first porphyrins (strictly, porphyrinogens, see below) are formed when four molecules of PBG condense together. The initial product of this reaction, catalysed by hydroxymethylbilane synthase (PBG deaminase) is hydroxymethylbilane. In the presence of uroporphyrinogen III cosynthase (also known as ‘uroporphyrinogen III synthase’), this is converted to uroporphyrinogen III. In the absence of this enzyme, hydroxymethylbilane is converted non-enzymatically to uroporphyrinogen I. A series of enzyme-catalysed reactions through isomers of the III series leads to the formation of protoporphyrin IXα. Haem is formed when iron is incorporated into this molecule in a reaction catalysed by ferrochelatase.
The porphyrinogens are themselves unstable and become oxidized to their corresponding porphyrins when they are excreted in faeces or urine. Porphyrinogens and porphyrin precursors are colourless. Porphyrins are dark red in colour and intensely fluorescent. The major sites of porphyrin synthesis are the liver and the erythroid bone marrow.
The rate-limiting step in this sequence of reactions is the first, catalysed by ALA synthase, which is susceptible to inhibition by the end product, haem.
These are a group of inherited diseases in which a partial deficiency of one of the enzymes of porphyrin synthesis leads to decreased formation of haem and thus, by releasing ALA synthase from inhibition, results in the formation of excessive quantities of porphyrin precursors (ALA and PBG) or porphyrins. When precursors are produced in excess, the clinical manifestations are primarily neurological (the precursors are neurotoxins). When porphyrins themselves are the major product, the predominant feature is photosensitivity: the porphyrins absorb light and become excited, inducing the formation of toxic free radicals. The porphyrias are diagnosed on the basis of their clinical features and the pattern of porphyrins and precursors present in blood and excreted in faeces and urine.
The porphyrias are classified as acute or non-acute, according to their clinical presentation, and hepatic or erythropoietic, depending on the major site of abnormal metabolism (Fig. 17.3). All the porphyrias are rare. Cutaneous hepatic porphyria (also known as porphyria cutanea tarda) is the most common but many cases are not inherited. Of the purely genetic types, acute intermittent porphyria is the most common, with a prevalence in the UK, where it occurs more frequently than in many countries, of 1–2 cases per 100 000 of the population. Unusually for inherited metabolic diseases, the mode of inheritance of most porphyrias is autosomal dominant, the exceptions being congenital erythropoietic porphyria, ALA dehydratase deficiency porphyria and hepatoerythropoietic porphyria (autosomal recessive). The features of the porphyrias are summarized in Figure 17.4. The genes for the enzymes involved in porphyrin synthesis have been identified and cloned, but the porphyrias are genetically heterogeneous; this hinders the application of molecular biological techniques to the identification of carriers and to screening for porphyrias.
Figure 17.3 Classification of the porphyrias. In addition to these conditions, two very rare porphyrias have been described: aminolaevulinic dehydratase deficiency porphyria (acute) and hepatoerythropoietic porphyria (chronic).
Figure 17.4 Features of the porphyrias. These conditions are caused by defects in each of the enzymes involved in haem synthesis (apart from the first one, δ-aminolaevulinic acid (ALA) synthase). The most important abnormalities are in bold; the changes shown for the acute porphyrias may only be present during an attack. AD, autosomal dominant; AR, autosomal recessive; N, neurological; P, photosensitizing. aType I isomers. bAD in some families.
Acute intermittent porphyria (AIP) is the commonest of these. Photosensitivity is never a feature of AIP, although it may occur in patients with hereditary coproporphyria and variegate porphyria.
These conditions are characterized by a tendency to acute attacks, separated by long periods of complete remission. The clinical features of acute attacks are summarized in Figure 17.5. Abdominal pain and psychiatric disturbances are nearly always present; peripheral neuropathy occurs in some 60% of patients. Attacks can be precipitated by various factors, including many drugs (see Fig. 17.5); most frequently implicated are barbiturates, oral contraceptives and alcohol. These probably act by increasing the activity of ALA synthase, in many cases by increasing the synthesis of hepatic cytochrome P450 and hence the demand for haem, thereby decreasing intrahepatic haem concentration and releasing the enzyme from inhibition. Some drugs, notably the sulphonamides, inhibit PBG deaminase directly. Whatever the cause, the resulting increased activity of the metabolic pathway increases the formation of metabolites before the enzyme block.
Figure 17.5 Clinical features and factors involved in acute attacks of porphyria; gastrointestinal, neuropsychiatric and cardiovascular derangements are all common.
Hormonal factors are also extremely important; symptoms rarely occur before puberty and may fluctuate in relation to menstruation or pregnancy. Women are affected more commonly than men. In some 90% of individuals who inherit the defective gene for AIP, the disease remains clinically latent throughout adult life.