CLINICAL PHARMACOLOGY 2003 (PART 30)

29 Cellular disorders and anaemias SYNOPSIS Rational use of haematinic drugs is essential to the correction of anaemia in its various forms The emergence of haemopoietic growth factors as drugs that stimulate erythroid or myeloid cell lines has opened the way to successful management of other forms of haematological disease Iron: therapy, acute overdose Vitamin B12 (cobalamins) Folic acid Haemopoietic growth factors Sickle cell anaemia Polycythaemia rubra vera Aplastic anaemia 'colouring matter' of the blood and the 'defective nature of the colouring matter' in anaemia were recognised In fact iron is essential not only to oxygen transport by red cells but as a catalyst for oxidative metabolism in all cells • Total body iron is 3-5 g (40-50 mg/kg) (male > female) • Haemoglobin contains about two-thirds of total body iron • Stores comprise about one-third (ferritin, a watersoluble protein-iron complex, and haemosiderin, an insoluble aggregate) in liver, marrow, spleen and muscle • 5-10% is present in tissues throughout the body in myoglobin, a variety of heme enzymes (e.g cytochromes) and non-haem enzymes (e.g metalloflavoproteins) Leukaemias and lymphomas: see Chapter 30 • Average Western diet contains 10-15 mg iron/day • Normal human absorbs 5-10% dietary iron, i.e 0.5-1.0 mg/d, which is adequate for an adult male or postmenopausal female but the menstruating or Iron pregnant woman requires 1-3 mg/d • Iron deficient or pregnant woman absorbs about 30% of dietary iron Iron, which was the metal symbolising strength in magical systems, used to be given to people suffering from weakness, and no doubt many were benefited, some psychologically (placebo reactors) and others because the weakness was due to iron deficiency anaemia The rational use of iron could not begin until both the presence of iron in the • Iron is lost from the body mainly in desquamated skin and gut cells and the daily loss in men is under I mg/day, in normal menstruating females 1.5 mg/day and in pregnancy averages mg/day • Menstrual loss is about 30 mg/period; menstruating women may therefore be in negative iron balance 587 29 CELLULAR DISORDERS AND ANAEMIAS IRON KINETICS Iron absorption takes place predominantly in the duodenum where the acid environment enhances solubility, but also throughout the gut, allowing sustained-release preparations to be used Most iron in food is present as ferric hydroxide, ferricprotein complexes or haem-protein complexes Ferrous (Fe++) iron is more readily absorbed than ferric (Fe+++) Thus the simultaneous ingestion of a reducing agent, such as ascorbic acid, increases the amount of the ferrous form; ascorbic acid 50 mg increases iron absorption from a meal by 2-3 times Food reduces iron absorption due to inhibition by phytates, tannates and phosphates Iron balance is determined by the difference between iron absorption and iron loss Humans lack a mechanism to excrete excess iron and physiological control of iron balance is achieved by regulation of absorption There is a reciprocal relationship between stores and absorption so that, as stores decline absorption increases and vice versa The mucosal cells of the proximal small bowel regulate iron absorption Dietary and administered iron is actively transported into the gut mucosal cell, probably involving a protein DMT1 though the precise details have not been established Two other proteins, hephaestin and ferroportin 1, appear to be involved in intracellular transport and release into the plasma respectively Regulation of absorption may involve one or more of: (1) control of mucosal uptake; (2) retention of iron in storage form in the mucosal cell and (3) transfer from the mucosal cell to the plasma Increased erythropoietic activity also stimulates increased absorption Iron that is not needed by the body may be bound to a protein (apoferritin) as ferritin and lost into the gut lumen when the mucosal cell is shed (2-3 days) Iron is eliminated at a near constant rate in the faeces of healthy people Iron that is required by the body forms a labile pool within the cell; if this pool is excessive it may stimulate production of more apoferritin in the mucosal cells to bind and lose more iron as ferritin when the cell is shed Labile pool iron in the Fe+++ form enters the plasma bound to a transport globulin, transferrin, which delivers it to the sites of physiological need, principally erythrocyte 588 precursors where it is used to form haem The major pathway of internal iron exchange is a unidirectional flow from plasma transferrin to the erythron (defined as all red cell elements at any stage of maturity), to the macrophage and back to plasma transferrin Over 80% of the iron passing through the transferrin compartment each day is flowing to and from the erythron Immature red cells acquire iron from transferrin through a specific transferrin receptor located on the cell membrane Within cells the iron regulatory proteins IRP-1 and IRP-2 control iron availability by translational control of the synthesis of transferrin receptor (increasing uptake) and of ferritin (increasing storage) There is a small amount of ferritin in the blood in balance with the iron stores Iron is stored as ferritin (which sequesters iron in a nontoxic but readily mobilised form) and its aggregate, haemosiderin, in the cells of the liver, bone marrow and spleen A measure of the state of iron stores is provided by the amount of ferritin in the serum (normally 20-300 mmol/1) and by the relationship of serum iron concentration (normally 10-30 mmol/1; reduced in iron deficiency) to the binding capacity of transferrin (normally 45-70 mmol/1; increased in iron deficiency) Ferritin is an acute-phase reactant and may be an inaccurate measure of iron stores in inflammatory states, e.g rheumatoid arthritis Recently developed techniques to measure the plasma level of soluble transferrin receptor (which is increased in iron deficiency but not by infection or inflammation) may help differentiate the anaemia of iron deficiency from that of chronic disease Prolonged heavy excess of iron intake overwhelms the mechanism described and results in haemosiderosis, as there is no physiological mechanism to increase iron excretion in the face of increased absorption Iron-deficient subjects absorb up to 20 times as much administered iron as those with normal stores Abnormalities of the small intestine may interfere with either the absorption of iron, as in coeliac disease and other malabsorption syndromes, or possibly with the conversion of iron into a soluble and reduced form, e.g following loss of acid secretion after a partial gastrectomy The formation of insoluble iron salts (such as phosphate and phytate) in the alkaline environ- I RON merit of most of the small intestine explains why much of the iron taken by mouth is not absorbed, even in severe iron deficiency Interactions Iron chelates in the gut with tetracyclines, penicillamine, methyldopa, levodopa, carbidopa, ciprofloxacin, norfloxacin and ofloxacin; it also forms stable complexes with thyroxine, captopril and biphosphonates These interactions can be clinically important Ingestion should be separated by hours Ascorbic acid increases absorption (see above) but its use (200 mg/day) is not clinically important in routine therapy; desferrioxamine binds iron and reduces absorption (see Poisoning, below); tea (tannins) and bran reduce absorption IRON THERAPY Iron therapy is indicated only for the prevention or cure of iron deficiency In general terms, making 25 mg of iron per day available to the bone marrow will allow an iron deficiency anaemia to respond with a rise of 1% of haemoglobin (0.15 g Hb/100 ml) per day; a reticulocyte response occurs between and 12 days An increase in the haemoglobin of at least g/dl after weeks of therapy is a reasonable criterion of an adequate response Oral preparations are the treatment of choice for almost all patients due to their effectiveness, safety and low cost Parenteral preparations should be restricted to the few patients unable to absorb or tolerate oral preparations Red cell transfusion is necessary only in patients with severe symptomatic anaemia or where chronic blood loss exceeds the possible rate of oral or parenteral replacement Oral iron therapy The goal of iron therapy is to repair the haemoglobin deficit and replenish storage iron When oral therapy is used it is reasonable to assume that about 30% of the iron will be absorbed and to give 180 mg of elemental iron daily for 1-3 months according to the degree of anaemia Iron stores are less easily replenished by oral therapy than by injection, and oral therapy (at lower dose) should be continued for 3-6 months after the haemoglobin concentration has returned to normal or until the serum ferritin exceeds 50 microgram/1 (or as long as blood loss continues) 29 Contraindications It is illogical to give iron in the anaemia of chronic infection where utilisation of iron stores is impaired; but such patients may also have true iron deficiency This may be difficult to diagnose without direct visualisation of stores in a bone marrow aspirate Iron should not be given in haemolytic anaemias unless there is also haemoglobinuria, for the iron from the lysed cells remains in the body Moreover the increased erythropoiesis associated with chronic haemolytic states stimulates increased iron absorption and adding to the iron load may cause haemosiderosis Iron therapy is needed in: • Iron deficiency due to dietary lack or to chronic blood loss • Pregnancy The extra iron required by mother and fetus totals 1000 mg, chiefly in the latter half of pregnancy The fetus takes iron from the mother even if she is iron deficient Dietary iron is seldom adequate and iron and folic acid (50-100 mg elemental iron plus folic acid 200-500 micrograms/day) should be given to pregnant women from the fourth month Opinions differ on whether all women should receive prophylaxis or only those who can be identified as needing it There are numerous formulations Parents should be particularly warned not to let children get at the tablets • Abnormalities of the gastrointestinal tract in which the proportion of dietary iron absorbed may be reduced, i.e in malabsorption syndromes such as coeliac disease • Premature babies, since they are born with low iron stores, and in babies weaned late There is very little iron in human milk and even less in cow's milk • Early treatment of severe pernicious anaemia with hydroxocobalamin, as the iron stores occasionally become exhausted by the surge in red cell formation Oral iron preparations There is an enormous variety of official and proprietary iron preparations For each milligram of elemental iron taken by mouth, ferrous sulphate is as effective as more expensive preparations It is particularly important to avoid initial overdosage with iron as the resulting symptoms may cause the patient to abandon 589 29 CELLULAR DISORDERS AND ANAEMIAS therapy A small dose may be given at first and increased after a few days The objective is to give 100-200 mg of elemental iron per day in an adult (3 mg/kg in a child) Iron given on a full stomach causes less gastrointestinal upset but less is absorbed than if given between meals; however, use with food is commonly preferred to improve compliance Commonly used preparations, given in divided doses, include: Ferrous Sulphate Tabs, 200-600 mg/d (providing 67-195 mg/d of elemental iron) Ferrous Gluconate Tabs, 300-1200 mg daily (providing 35-140 mg/d of elemental iron) Ferrous Furmarate Tabs, 200-600 mg daily (providing 130-195 mg/d of elemental iron) Ferrous sucdnate and ferrous glycine sulphate are alternatives Choice of oral iron preparation Oral iron is used both for therapy and for prophylaxis (pregnancy) of anaemia in people who are often feeling little if any ill-health Because of this, the occurrence of gastrointestinal upset is particularly important as it may cause the patient to give up taking iron The evidence as to which preparation provides best iron absorption with least adverse effects is conflicting Gastrointestinal upset is minimal if the daily dose does not exceed 180 mg elemental iron and if iron is given with food A suggested course Start a patient on ferrous sulphate taken on a full stomach once, then twice, then thrice a day If gut intolerance occurs, stop the iron and reintroduce it with one week for each step If this seems to cause gastrointestinal upset, try ferrous gluconate, succinate or fumarate If simple preparations (above) are unsuccessful, and this is unlikely, then the pharmaceutically sophisticated and expensive sustained-release preparations may be tried They release iron slowly and only after passing the pylorus, from resins, chelates (sodium iron edetate) or plastic matrices, e.g Slow-Fe, Ferrograd, Feospan, so that iron is released in the lower rather than the upper small intestine Patients who cannot tolerate standard forms even when taken with food may get as much iron with fewer unpleasant symptoms if they use a sustained-release formulation Liquid formulations are available for adults who prefer them and for small children, e.g Ferrous 590 Sulphate Oral Solution, Paediatric: ml contains 12 mg of elemental iron: but they stain the teeth Polysaccharide-iron complex (Niferex): ml contains 100 mg of elemental iron There are numerous other iron preparations which can give satisfactory results Sustained-release and chelated forms of iron (see above) have the advantage that poisoning is less serious if a mother's supply is consumed by young children, a real hazard Iron therapy blackens the faeces but does not generally interfere with modern tests for occult blood (commonly needed in investigation of anaemia), though it may give a false positive with some older occult blood tests, e.g guaiac test Failure of oral iron therapy is most commonly due to poor patient compliance, persistent bleeding and, as with all drug therapy, wrong diagnosis Adverse effects Most patients tolerate oral iron therapy but 10-20% have symptoms that may be attributed to iron, generally gastrointestinal upset These effects of oral iron include nausea, abdominal pain, and either constipation or diarrhoea Upper GI effects appear to be dose-related and are best managed by ingestion of the tablet with or after food and/or reduction in the amount of iron content in each dose This will prolong the necessary period of treatment Diarrhoea or constipation can usually be treated symptomatically without a change in regimen Parenteral iron therapy This may be required if: • Iron cannot be absorbed from the intestine • The patient cannot be relied on to take it or experiences intolerable gut symptoms Speed of haemopoietic response is not quicker than that with full doses of oral iron reliably taken and normally absorbed, for both provide as much iron as an active marrow can use, but a course of injected iron is stored and utilised over months The ionised salts of iron given orally are unsuitable as parenteral preparations as they are powerful protein precipitants and un-ionised iron complexes are used RON 29 Intramuscular iron Iron sorbitol inj (50 mg of iron/ml) is an iron-sorbitol-citric-acid complex of MW < 5000 that is rapidly absorbed into the blood from the site of i.m injection Iron sorbitol is bound to plasma globulin, transferrin, and is stored in the marrow and liver It is not substantially taken up in the reticuloendothelial system Excess unbound iron is excreted in the urine (about 30% of the dose) which may turn black transiently at the time of peak iron excretion or only on standing for some hours iron and folic acid, the lack of the latter may not be obvious because haematopoiesis is impaired by insufficiency of iron If iron is supplied increased erythropoiesis reveals the folic acid deficiency This is most likely to happen in pregnancy due to high fetal requirements for both haematinics and so folic acid is commonly given to all pregnant patients with anaemia (see below); it also occurs in malabsorption syndromes where both may be malabsorbed Intravenous iron Iron dextran inj (ferric hydroxide complexed with dextrans; 50 mg/ml) and iron sucrose inj (ferric hydroxide complexed with sucrose; 20 mg/ml) are administered by slow i.v injection or infusion (not recommended for children) Oral iron therapy should not be given 24 h before i.m injections begin and for days after the last i.v injection; not only is continuation unnecessary, but it may promote adverse reactions by saturating the plasma protein (transferrin) binding capacity so that the injected iron gives a higher unbound plasma iron concentration than is safe Acute overdose: poisoning Doses The approximate total requirement is ascertained from manufacturers' dosage schedules which relate body weight to the haemoglobin deficit Iron sorbitol is normally given daily or on alternate days where tolerance is low It is given by deep i.m injection, which can be painful It stains the skin (for up to years) but this can be minimised by inserting the needle through the skin and then moving the skin and subcutaneous tissue laterally before entering the muscle so that the needle track becomes angulated when the needle is withdrawn (the Z-technique) Adverse effects General reactions include headache, dizziness, nausea, vomiting, disorientation, pressure sensations in the chest, myalgia, hypotension, a metallic taste, urticaria and hypersensitivity Intravenous iron may rarely cause anaphylactoid reactions and facilities for cardiopulmonary resuscitation should be available Folic acid deficiency may be unmasked by effective iron therapy Where there is a deficiency of both High doses of iron salts by mouth can cause severe gastrointestinal irritation and even necrosis of the mucous membrane Autopsy shows severe damage to brain and liver Iron poisoning is particularly dangerous in children Sustained-release forms are safer in homes where heedless parents live with small children Ferrous sulphate is the most toxic Typically acute oral iron poisoning has the following phases: 0.5-1 h after ingestion there is abdominal pain, grey/black vomit, diarrhoea, leucocytosis and hyperglycaemia Severe cases are indicated by acidosis and cardiovascular collapse which may proceed to coma and death There follows a period of improvement lasting 6-12 h, which may be sustained or which may deteriorate to the next stage Jaundice, hypoglycaemia, bleeding, encephalopathy, metabolic acidosis and convulsions are followed by cardiovascular collapse, coma and sometimes death 48-60 h after ingestion 1-2 months later, upper gastrointestinal obstruction may result from scarring and stricture Treatment of acute iron poisoning is urgent and immediate efforts must be made to chelate iron in the blood and in the stomach and intestine Raw egg and milk help to bind iron until a chelating agent is available The first step should be to give desferrioxamine 1-2 g i.m.; the dose is the same in adults and children Only after this should gastric aspiration or emesis be performed If lavage is used, the water should contain desferrioxamine g/1 After empty591 29 CELLULAR DISORDERS AND ANAEMIAS ing the stomach, desferrioxamine 10 g in 50-100 ml water should be left in the stomach to chelate any remaining iron in the intestinal lumen; it is not absorbed Subsequently, desferrioxamine should be administered by i.v infusion not exceeding 15 mg/kg/h (maximum 80 mg/kg/24 h) or further i.m injections (2 g in sterile water 10 ml) should be given 12-hourly Poisoning is severe if the plasma iron concentration exceeds the total iron binding capacity (upper limit 75 mmol/1) or the plasma becomes pink due to the large formation of ferrioxamine (see below) If severe poisoning is suspected i.v rather than i.m administration of desferrioxamine is indicated without waiting for the result of the plasma concentration Desferrioxamine (deferoxamine) (Desferal) (t1/2 h) is an iron-chelating agent (see Chelating agents, p 154) During a systematic investigation of actinomycete metabolites, iron-containing substances (sideramines) were discovered One of these substances was ferrioxamine The iron in this can be removed chemically, leaving desferrioxamine When desferrioxamine comes into contact with ferric iron, its straight-chain molecule twines around it and forms a nontoxic complex of great stability (ferrioxamine), which is excreted in the urine giving it a red/orange colour, and in the bile It is not absorbed from the gut and must be injected for systemic effect In acute poisoning, as opposed to chronic overload, desferrioxamine g chelates the iron contained in about 10 tablets of ferrous sulphate or gluconate It has a negligible affinity for other metals in the presence of iron excess Desferrioxamine has been shown to be effective in the therapy of acute iron poisoning and in the treatment and perhaps in the diagnosis of diseases associated with chronic iron accumulation A topical formulation is available for ocular siderosis Serious adverse effects are uncommon but include rashes and anaphylactic reactions; with chronic use cataract, retinal damage and deafness can occur Hypotension occurs if desferrioxamine is infused too rapidly and there is danger of (potentially fatal) adult respiratory distress syndrome if infusion proceeds beyond 24 h.1 592 Chronic iron overload Humans are uniquely unable to excrete excess iron so that, if there is uncontrolled iron intake, it progressively accumulates Grossly excessive parenteral iron therapy or a hundred or more blood transfusions (as in treatment of thalassaemia2) can lead to haemosiderosis Oral iron therapy over many years has also been reported to cause haemosiderosis Treatment of chronic iron overload, e.g haemochromatosis, patients who are transfusion-dependent due to chronic haemolytic anaemias, thalassaemia and refractory anaemias with transfusional iron overload (siderosis) The goal of therapy is the reduction and maintenance of body iron stores at normal or near-normal levels to avoid the tissue damage associated with iron overload Iron may be removed by repeated venesection in haemochromatosis where there is no anaemia A single vensection of 450 ml of blood, in the absence of anaemia, removes 200-250 mg of iron and can be repeated weekly in individuals with haemochromatosis until the ferritin reaches the normal range After complete removal of the iron load, maintenance therapy in the form of venesection every 3-4 months is required A small number of patients with haemosiderosis and cardiac failure may require chelator therapy Patients with transfusion siderosis require a long-term programme of chelation therapy In patients who are transfusion-dependent from infancy (thalassaemia major, congenital refractory anaemia) chelation therapy is commenced after 10-20 transfusions at about years of age In older patients with acquired transfusion-dependent anaemias chelation is commenced after 20 transfusions or when the serum ferritin is 2-3 times the upper limit of normal Chelation can be effectively carried out only by slow parenteral administration of desferrioxamine s.c or i.v through an indwelling catheter with a small portable syringe pump e.g over 9-12 h Tenenbein M et al 1992 Lancet 339: 699 A 26-year-old subject with beta-thalassaemia major had been transfused 404 units of blood over his lifetime His iron stores were so high (estimated at above 100 g) that he triggered a metal detector at an airport security checkpoint (Jim R T S 1979 Lancet 2: 1028) VITAM I N nocturnally on nights per week Simultaneous oral administration of ascorbic acid is to be avoided; it increases the availability of free iron for chelation but carries the risk of mobilising iron from relatively safe reticuloendothelial storage sites to a potentially toxic pool in parenchymal cells This regimen can put a transfusion-dependent patient into the desired negative iron balance Compliance is often a problem and is typically difficult during teenage years in those with lifelong transfusiondependence The expense of chelation therapy over a long period is currently enormous and raises serious ethical problems in economically poor countries where most patients with thalassaemia and haemoglobinopathies live A safe, effective, inexpensive, orally-absorbed iron chelating agent would improve compliance and the quality of life of affected patients Deferiprone, which is the best of many agents examined, is less effective than desferrioxamine, carries a risk of agranulocytosis and may itself cause tissue fibrosis It remains under clinical trial but may be too toxic for general use Vitamin B 12 PERNICIOUS ANAEMIA In 1925, it was demonstrated that two factors were required to cure pernicious anaemia: one in the food (extrinsic factor) and one in gastric juice (intrinsic factor) • Extrinsic factor, cyanocobalamin (vitamin B12), was isolated in 1948 • Intrinsic factor (a glycoprotein secreted by the parietal cells of the fundus and cardia) acts solely as a vehicle for carrying the important extrinsic factor into the body via receptors in the ileum COBALAMINS Cobalamins comprise a family of compounds which share a complex structure Vitamin B]2 is known as cyanocobalamin because when originally isolated, an in-vitro artefact had placed a cyan group in the cobalt P position Vitamin B12 is an active cellular coenzyme essential for demethy- B 29 lation of tetrahydrofolate and thus for DNA synthesis Animals cannot synthesise cobalamin and so are directly or indirectly dependent upon microorganisms for it Cobalamin is produced in nature only by cobalamin-producing microorganisms, and herbivores obtain their supply from plants contaminated with bacteria and faeces Carnivores obtain their supply by ingesting the muscular and parenchymal tissues of these animals Animal protein is the major dietary source of cobalamin in man Although bacteria in the human colon synthesise cobalamin, it is formed too distally for absorption by the ileal transport system Rabbits in the wild would suffer from B12 deficiency if they did not eat their own faeces In the presence of intrinsic factor about 70% of ingested cobalamin is absorbed, in its absence < 2% is absorbed Some cyanocobalamin may be absorbed by passive diffusion, i.e independently of intrinsic factor, though less reliably and only with large doses Dietary deficiency is virtually confined to people too impoverished to buy meat, and to Vegans, a sect of particularly uncompromising vegetarians Deficiency of vitamin B12 in the body leads to: • Megaloblastic anaemia • Degeneration of the brain, spinal cord (subacute combined degeneration) and peripheral nerves; symptoms may be psychiatric and physical • Abnormalities of epithelial tissue, particularly of the alimentary tract, e.g sore tongue and malabsorption ABSORPTION AND TRANSPORT The daily requirement of cobalamin is about 3.0 micrograms Absorption takes place mainly in the terminal ileum, and it is carried in plasma bound to proteins Some 90% of recently absorbed or administered cobalamin is carried on transcobalamin II an important transport protein which is rapidly cleared from the circulation (t1/2 6-9 minutes) Hereditary deficiency of transcobalamin II causes severe cobalamin deficiency About 80% of all circulating cobalamin is bound to transcobalamin I (t1/2 9-12 days) which is possibly a plasma storage form (hereditary deficiency of which is of no consequence) Cobalamin in its reduced form 593 29 CELLULAR DISORDERS AND ANAEMIAS cob(I)alamin functions as a coenzyme for methionine synthase in a reaction that generates tetrahydrofolate, and is critical for DNA and RNA synthessis Cobalamin is not significantly metabolised and passes into the bile (there is enterohepatic circulation which can be interrupted by intestinal disease and hastens the onset of clinical deficiency), and is excreted via the kidney Body stores amount to about mg (mainly in the liver) and are sufficient for 2-4 years if absorption ceases INDICATIONS FORVITAMIN B 12 Indications for administration are the prevention and cure of conditions due to its deficiency Hydroxocobalamin is preferred for clinical use Pernicious (Addisonian) anaemia The atrophic gastric mucosa is unable to produce intrinsic factor (and acid) due to an autoimmune reaction to gastric parietal cells and intrinsic factor itself, there is failure to absorb vitamin B12 in the terminal ileum so that deficiency results Despite its name (given when no treatment was known and it was believed to be a neoplastic disorder due to the appearance of the megaloblastic bone marrow), the prognosis of a patient with uncomplicated pernicious anaemia, properly treated with hydroxocobalamin, is little different from that of the rest of the population The neurological complications, particuarly spasticity, develop only after prolonged severe deficiency but may be permanent; they are rarely seen today Total removal of the stomach or atrophy of the mucous membrane in a postgastrectomy remnant may, after several years, lead to a similar anaemia Malabsorption syndromes In stagnant loop syndrome (bacterial overgrowth which competes for the available cobalamin and can be remedied by a broad-spectrum antimicrobial), ileal resection, Crohn's disease and chronic tropical sprue affecting the terminal ileum, vitamin B12 deficiency is common although megaloblastic anaemia occurs only relatively late The fish tape worm Diphyllobothrum latum which can infest humans who eat raw or partially cooked freshwater fish roe can grow up to 10 meters in the gut and competes for ingested cobalamin 594 Tobacco amblyopia has been attributed to cyanide intoxication from strong tobacco which interferes with the coenzyme function of vitamin B12; hydroxocobalamin (not cyanocobalamin) may be given DIAGNOSIS OF B|2 DEFICIENCY The serum concentration of vitamin B12 is low (normal 170-925 nanogram/1) In severe deficiency there is pancytopenia, the blood film shows anisopoikilocytosis with oval macrocytes and hypersegmented neutrophils; the marrow is megaloblastic In many patients with pernicious anaemia antibodies to intrinsic factor can be identified in the serum Absorption of radioactive vitamin B12 (Schilling test) helps to distinguish between gastric and intestinal causes First: the patient is given a small dose of radioactive vitamin B12 orally, with a simultaneous large dose of nonradioactive vitamin B12 intramuscularly The large injected dose saturates binding sites so that any of the oral radioactive dose that is absorbed cannot bind and will be eliminated in the urine where it can easily be measured (normally > 10% of the administered dose appears in urine collected for 24 h, if renal function is normal) In pernicious anaemia and in malabsorption, gut absorption and therefore subsequent appearance of radioactivity in the plasma (measured 8-12 h later) and urine are negligible Second: the test is repeated with intrinsic factor added to the oral dose The radioactive vitamin B12 is now absorbed in pernicious anaemia (but not in intestinal malabsorption) and is detected in plasma and urine Both stages of the test are needed to maximise reliability of diagnosis of pernicious anaemia CONTRAINDICATIONSTOVITAMIN B|2 Inconclusively diagnosed anaemia is an important contraindication Therapy of pernicious anaemia must be both adequate and lifelong, so that accurate diagnosis is essential Even a single dose of vitamin B12 interferes with the haematological picture for weeks (megaloblastic haematopoiesis reverts to normal within 12 hours), although the Schilling test remains diagnostic FOLIC ACID (PTEROYLG LUTAM 1C ACID) PREPARATIONS AND USE Hydroxocobalamin is bound to plasma protein to a greater extent than is cyanocobalamin, with the result that there is less free to be excreted in the urine after an injection and rather lower doses at longer intervals are adequate Thus hydroxocobalamin is preferred to cyanocobalamin, though the latter can give satisfactory results as the doses administered are much greater than are required physiologically Cyanocobalamin remains available The initial dose in cobalamin deficiency anaemias, including uncomplicated pernicious anaemia, is hydroxocobalamin mg i.m every 2-3 days for doses to induce remission and to replenish stores Maintenance may be mg every months; higher doses will not find binding sites and will be eliminated in the urine Higher doses are justified during renal or peritoneal dialysis where hydroxycobalamin clearance is increased, and resultant raised plasma methylmalonic acid and homocysteine represent an independent risk factor for vascular events in these patients (see later) Routine low dose supplements of hydroxycobalamin, folate and pyridoxine fail to control hyperhomocysteinaemia in 75% of dialysis patients but supraphysiological doses are effective: hydroxycobalamin mg/d, folic acid 15 mg/d and pyridoxine 100 mg/d After initiation of therapy, patients feel better in days, reticulocytes peak at 5-7 days and the haemoglobin, red cell count and haematocrit rise by the end of the first week These indices normalise within months irrespective of the starting level Failure to respond implies a wrong or incomplete diagnosis (coexistent deficiency of another haematinic) The initial stimulation of haemoglobin synthesis often depletes the iron and folate stores and supplements of these may be needed Hypokalaemia may occur at the height of the erythrocyte response in severe cases It is attributed to uptake of potassium by the rapidly increasing erythron (erythrocyte mass) Oral potassium should be given prior to initiating therapy in a patient with low or borderline potassiuim levels Once alternative or additional causes of the anaemia have been excluded, inadequate response should be treated by increased frequency of injections as well as increased amount (because of 29 urinary loss with high plasma concentrations) The reversal of neurological damage is slow (and rarely marked) and the degree of functional recovery is inversely related to the extent and duration of symptoms Haemoglobin estimations are necessary at least every months to check adequacy of therapy and for early detection of iron deficiency anaemia due to achlorhydria (common in patients with pernicious anaemia > 60 years) or carcinoma of the stomach, which occurs in about 5% of patients with pernicious anaemia When injections are refused or are impracticable (rare allergy, bleeding disorder), administration as snuff or aerosol has been effective, but these routes are less reliable Large daily oral doses (1000 micrograms) are probably preferable; depleted stores must be replaced by parenteral cobalamin before switching to the oral preparation; the patient must be compliant; monitoring of the blood must be more frequent and adequate serum vitamin B12 levels must be demonstrated Adverse effects virtually not occur, but use of vitamin B12 as a 'tonic' is an abuse of a powerful remedy for it may obscure the diagnosis of pernicious anaemia, which is a matter of great importance in a disease requiring lifelong therapy and prone to serious neurological complications The latter danger is of particular significance when a megaloblastic anaemia due to pernicious anaemia is incorrectly diagnosed as due to folate deficiency; here folic acid, if used alone (see below) may accelerate progression of subacute combined degeneration of the nervous system Folk acid (pteroylglutamic acid) Folic acid3 was so named because it was discovered as a bacterial growth factor present in spinach leaves It is one of the B group of vitamins and was soon shown to be the same substance as that Latin: folium, a leaf 595 29 CELLULAR DISORDERS AND ANAEMIAS present in yeast and liver which cured a nutritional macrocytic anaemia in Indian women FUNCTIONS Folic acid is itself inactive; it is converted into the biologically active coenzyme, tetrahydrofolic acid, which is important in the biosynthesis of amino acids and DNA and therefore in cell division The formyl derivative of tetrahydrofolic acid is folinic acid and this is used to bypass the block when the body fails to effect the conversion of folic acid (see Folic acid antagonists, p 606) Ascorbic acid protects the active tetrahydrofolic acid from oxidation; the anaemia of scurvy, although usually normoblastic, may be megaloblastic due to deficiency of tetrahydrofolic acid Deficiency of folic acid leads to a megaloblastic anaemia because it is necessary for the production of purines and pyrimidines, which are essential precursors of deoxyribonucleic acid (DNA) The megaloblastic marrow of cobalamin deficiency is due to interference with folic acid utilisation and the morphological changes of cobalamin deficiency can be reversed by folic acid It is vital to realise that folic acid does not provide adequate treatment for pernicious anaemia Nor does vitamin B12 provide adequate treatment for the megaloblastic anaemia of folic acid deficiency, although a partial response may occur because vitamin B12 plays a role in folate metabolism OCCURRENCE AND REQUIREMENTS Folic acid is widely distributed, especially in green vegetables, yeast and liver Daily requirement of folic acid in an adult is some 50-100 micrograms and a diet containing 400 micrograms of polyglutamates will provide this In childhood the requirement is 50 micrograms per day about x more on a weight-for-weight basis Body stores last about months Dietary deficiency Folate deficiency is extremely common in the setting of general malnutrition in developing countries and is a particular problem in childhood In Western countries folate deficiency occurs in alcoholics, some slimming diets, the elderly, the infirm and psychiatric patients Pregnancy Folic acid requirement is increased to 300-400 microgram a day This cannot be met from the diet by one-third of women in Western societies and the problem is greater in less economically developed countries where nutritional deficiency may be aggravated by high red cell turnover due to haemoglobinopathies and endemic malaria For this reason folic acid is added to iron for prophylaxis of anaemia in pregnancy The dose needed is about 300 micrograms of folic acid a day, which is insufficient to alter the blood picture of pernicious anaemia and so there is no risk of masking that disease (pernicious anaemia is also very rare in women of reproductive age and is probably incompatible with a successful pregnancy) A large number of preparations of iron with folic acid is available (see also Iron therapy, p 591) They are suitable only for prevention Larger doses may be used in therapy of anaemia during pregnancy (see below); it will remit spontaneously some weeks after delivery Vigorous iron therapy in pregnancy may unmask a folate deficiency During lactation requirements remain increased Prevention of fetal neural tube defect (spina bifida) Folic acid supplementation taken before conception and during the early weeks of pregnancy has been shown in an 8-year trial to prevent the condition in pregnancies subsequent to an affected birth.4 Women hoping to conceive and who have had an affected child are advised to take folic acid mg/day To prevent a first occurrence 400 micrograms/day should be taken both before conception, or as soon as possible after diagnosis.5 In both cases folate supplement should be taken for the first 12 weeks of pregnancy INDICATIONS Folic acid is used to prevent or cure deficiency of folate which are due either to a decreased supply or to an increased requirement 596 MRC Vitamin Study research group 1991 Lancet 338:131 A supplement of folic acid mg/day is proposed for fuller risk reduction Wald N J, Law M R, Morris J K et al 2001 Quantifying the effect of folic acid Lancet 358: 2069-2073 HAEMOPOIETIC Premature infants Supplementation is needed because these infants miss the build-up of folate stores that normally occurs in the last few weeks of pregnancy Malabsorption syndromes Particularly in glutensensitive enteropathy and tropical sprue, poor absorption of folic acid from the small intestine often leads to a megaloblastic anaemia Drugs Antiepilepsy drugs, particularly phenytoin, primidone and phenobarbital, occasionally cause a macrocytic anaemia that responds to folic acid This may be due to enzyme induction by the antiepileptics increasing the need for folic acid to perform hydroxylation reactions (see Epilepsy) but other factors such as reduced absorption may be involved Administration of folic acid causes a recurrence of seizures in some patients Some antimalarials, e.g pyrimethamine, may interfere with conversion of folates to the active tetrahydrofolic acid, causing macrocytic anaemia Methotrexate, another folate antagonist, may cause a megaloblastic anaemia especially when used long-term for leukaemia, rheumatoid arthritis or psoriasis Miscellaneous causes of excess utilisation or loss In chronic haemolytic states, where erythropoiesis is accelerated, and in myelofibrosis, where haemopoiesis is inefficient, folate requirement is increased Extensive shedding of skin cells in exfoliative dermatitis, inflammatory states, e.g rheumatoid arthritis, and malignant disease (lymphoma), can similarly lead to folate deficiency Folate loss during chronic haemodialysis may be sufficient to require replacement CONTRAINDICATIONS Imprecisely diagnosed megaloblastic anaemia is the principal contraindication Tumour cell proliferation in some cancers may be folate dependent and folic acid should be used in malignant disease only where there is confirmed folate deficiency anaemia PREPARATIONS AND DOSAGE Synthetic folic acid is taken orally; for therapy mg daily is usually given for months, or indefinitely if GROWTH FACTORS 29 the cause of deficiency cannot be removed; 15 mg/day may be needed in malabsorption states though usually mg is adequate There is no advantage in giving folinic acid instead of folic acid, except in the treatment of the toxic effects of folic acid antagonists such as methotrexate (folinic acid 'rescue', see p 608) • For prophylaxis, with iron, in pregnancy, see page 589 • For prophylaxis in haemolytic diseases and in renal dialysis: mg per day or per week depending on need Adverse reactions are rare: allergy occurs, and status epilepticus may be precipitated Haemopoietic growth factors Cloning of growth factor genes and recombinant DNA technology allow the large-scale production of cytokines for clinical use Growth factors are now available to stimulate both erythroid and myeloid cell lines These factors are potentially useful whenever there is cytopenia, whether due to disease or to cytotoxic chemotherapy ERYTHROPOIETIN Erythropoietin is a glycoprotein hormone encoded by a gene on the long arm of chromosome (7q) and 90% is produced in the kidney (the remainder in the liver and other sites) in response to hypoxia The anaemia of chronic renal failure is largely due to failure of the diseased kidneys to make enough erythropoietin The principal action of the hormone is to stimulate the proliferation, survival and differentiation of erythrocyte precursors The manufacture of erythropoietin for clinical use became possible when the human gene was successfully inserted into cultured hamster ovary cells Epoetin (recombinant derived human erythropoietin) must be given s.c (which may be more effective) or i.v.; the t1/2 is h and appears not to be affected by dialysis Maximum reticulocyte response 597 29 CELLULAR DISORDERS AND ANAEMIAS occurs in days Self-administration at home three times a week is practicable; the dose is adjusted by response Iron reserves must be adequate for optimum erythropoiesis, i.e serum ferritin should exceed 100 micrograms/1 Epoetin is available as two preparations, epoetin alpha and epoetin beta, which are interchangeable Epoetin is effective in the anaemia of chronic renal failure to an extent that it significantly enhances the patients' quality of life Patients become independent of blood transfusion, with great benefit to blood transfusion services as well as to themselves Recombinant erythropoietin has also been used in anaemia of rheumatoid arthritis, prematurity, following cancer chemotherapy, myelodysplasia and zidovudine-treated AIDS, and to improve the quality of presurgical autologous blood collection Athletes in track events and cycling seeking advantage through increased haemoglobin concentrations have misused it Adverse effects A dose-dependent increase in arterial blood pressure follows the rise in red cell mass and encephalopathy may occur in some previously hypertensive patients Arteriovenous shunts of dialysis patients, especially those that are compromised, may thrombose as a result of increased blood viscosity Iron deficiency may occur, as increased haematopoiesis outstrips available iron stores, and this can be a cause of inadequate response to the hormone; parenteral iron therapy may be needed Transient influenza-like symptoms may accompany the first i.v injections COLONY-STIMULATING FACTORS A number of cytokines (see p 280) stimulate the growth, differentiation and functional activity of myeloid progenitor cells As the name implies the function of these polypeptides was defined by invitro colony assays of bone marrow progenitors They have effects on all myeloid cells including the multipotential stem cells (but probably not the more immature pluripotential cells), intermediate progenitors and circulating mature cells Those in clinical use are described below 598 Granulocyte colony-stimulating factors: G-CSF, an 18 kDa protein encoded by a gene on the long arm of chromosome 17 (17q), stimulates the proliferation of granulocyte progenitors and activates neutrophil function Filgrastim is recombinant human granulocyte colony-stimulating factor A single dose will cause the neutrophil count to rise 4-5-fold within hours and the increased count persists up to 72 h The drug is rapidly cleared after i.v injection (t1/2 2h) and administration by i.v infusion or s.c is necessary to prolong plasma concentration High concentrations are found in plasma, bone marrow and kidneys It is degraded to its component amino acids G-CSF is widely used to mobilise bone marrow stem cells into the peripheral blood to support both autologous and allogeneic peripheral blood progenitor transplantation The use of peripheral blood progenitors as opposed to bone marrow progenitors is associated with earlier neutrophil and platelet recovery, fewer red cell transfusions and earlier discharge from hospital Another major use of G-CSF is for patients with neutropenia as a result of cytotoxic chemotherapy, to shorten the duration of neutropenia and reduce morbidity due to infection It is also used for the same purpose after autologous and allogeneic bone marrow transplantation, in aplastic anaemia, AIDS, and congenital, cyclical and idiopathic neutropenia In combination with epoetin, G-CSF can be effective in the management of some patients with myelodysplastic syndromes G-CSF not only improves the neutrophil count, but dramatically improves the proportion of patients with a raised haemoglobin in response to epoetin possibly by reduction of erythroid apoptosis (the cause of ineffective erythropoiesis) Adverse effect Medullary bone pain occurs with high i.v doses Musculoskeletal pain, dysuria, splenomegaly, allergic reactions and abnormality of liver enzymes also occur Lenograstim is similar Granulocyte-monocyte colony-stimulating factor: GM-CSF, a glycoprotein of 14-35 kDa encoded by a gene on the long arm of chromosome (5q), has a broader spectrum of activity than G-CSF, POLYCYTHAEM IA RUBRAVERA stimulating both monocyte and granulocyte production with functional effects on the mature cells of both cell lines Molgramostim (recombinant human granulocytemonocyte colony-stimulating factor) has a t1/2 of h and administration by i.v infusion or s.c is needed to maintain plasma concentration Molgramostim has also been used to mobilise peripheral blood progenitors and to reduce cytotoxic-induced neutropenia, and in bone marrow transplantation and aplastic anaemia It is now less widely used than G-CSF Molgramostim has also been used for neutropenia caused by ganciclovir and for AIDSrelated cytomegalovirus retinitis It appears to be synergistic with amphotericin in the treatment of invasive pulmonary aspergillosis possibly by activation of macrophages to enhance killing of phagocytosed fungi Adverse effects Molgramostim causes medullary bone pain, skin rashes, lethargy and myalgia in 1020% of patients It may also cause fever, the interpretation of which presents a clinical dilemma in neutropenic patients who are subject to sepsis Pleural and pericardial effusions occur after high doses Thrombopoietin (TPO), a 36 kDa protein encoded by a gene on the long arm of chromosome (3q) stimulates the growth and differentiation of megakaryocyte progenitors, mature megakaryocytes and primes platlets to respond to stimuli Recombinant human TPO has been examined in a small number of clinical trials and found to produce a dosedependent increase in bone marrow megakaryocytes and the peripheral blood platelet count If it proves nontoxic (concerns include the potential for platelet activation leading to thrombosis, and the risk of myelofibrosis), it may have a role in the treatment of chemotherapy-induced thrombocytopenia Hydroxyurea (hydroxycarbamide) in sickle cell anaemia In sickle cell disease, haemoglobin S (HbS) when deoxygenated forms polymers which result in the 29 red cells changing from flexible biconcave discs to unyielding sickle shapes that obstruct blood flow This gives rise to the clinical features of haemolysis with shortened red cell survival, anaemia and painful bone crises Haemoglobin F (HbF) interferes with the polymerisation process and is protective against the disease Hydroxyurea (hydroxycarbamide) is the first widely available and affordable agent that provides real benefit It acts by perturbing the maturation of erythrocytes and promoting HbF production The mode of action may be more complex; reduction in leukocyte counts may reduce vaso-occlusive events; reduced red cell and endothelial adhesiveness may be a direct effect Beneficial effects have been seen in adults, children and infants Long-term hydroxyurea (hydroxycarbamide) (at close to myelotoxic doses) raises HbF to 15-20% and reduces the frequency of hospitalisation, pain, acute chest syndrome and blood transfusion Neurological complications e.g stroke, may not be reduced Some 10-20% of patients will fail to respond due to the condition of the bone marrow, or genetic effects (see also p 607, 613) Adverse effects The long-term risk of leukaemogenesis cannot yet be assessed There appears to be no adverse effects on growth or development Polycythaemia rubra vera The clinical course of polycythaemia rubra vera (PRV) is marked by a high risk of thrombotic complications and a variable incidence of transformation to myelofibrosis or acute myeloblastic leukaemia The object of treatment is to minimise the risk of thrombosis and to prevent transformation The following are used Phlebotomy The object is to reduce the haematocrit to less than 0.45 by venesection (300-500 ml) every days Thereafter the attempt is made to maintain normal status by occasional venesection Iron deficiency may occur and need treatment although this may result in a need for more frequent venesection Additional myelosuppressive therapy is required in most patients This is indicated if frequent vene599 29 CELLULAR DISORDERS AND ANAEMIAS section is required to maintain a normal haematocrit or if the platelet count continues high (added risk of thrombosis) Radiophosphorus (32P, sodium radiophosphate) is given i.v Phosphorus is concentrated in bone and in cells that are dividing rapidly, so that the erythrocyte precursors in the bone marrow receive most of the [3-irradiation The effects are similar to those of whole-body irradiation, and in PRV, 32P is a treatment option for those > 65 years (accumulation in the gonads precludes its use in younger patients) The maximum effect on the blood count is delayed 1-2 months after a single dose that usually provides control for 1-2 years It reduces vascular events and delays progression to myelofibrosis Excessive depression of the bone marrow including leucocytes and platelets is the main adverse effect, but is seldom serious Acute myeloid leukaemia occurs more frequently in patients treated with 32P especially when used in combination with hydroxyurea Alkylating agents Busulfan is a radio-mimetic cytotoxic agent that is effective in PRV, reducing vascular events and delaying myelofibrosis Its mutagenic potential should restrict its use to older patients Chlorambucil and combination chemotherapy should be avoided because of excessive leukaemogenic risk Hydroxyurea (hydroxycarbamide) This antimetabolite is thought to carry a lower risk of leukaemogenesis than either of the above agents but anxieties remain It effectively reduces the incidence of thrombosis and is regarded as more acceptable therapy for younger patients Anagrelide is an oral agent which inhibits platelet aggregation but at lower doses it lowers platelet counts in man due to a marked effect on megakaryocyte maturation It is nonmutagenic and effectively controls thrombocytosis in PRV and essential thrombocythaemia (ET) Adverse effects are cardiovascular: headache, forceful heartbeats, fluid retention and arrhythmias Interferon alfa is another probably non-leukaemogenic alternative for younger patients Other features Pruritus is troublesome and difficult to relieve; it may be helped by Ha- and 600 H2-histamine receptor blockers alone or together Hyperuricaemia, due to cell destruction, is prevented by allopurinol; and iron and folate deficiency by replacement doses (due to the rapid response of the myeloproliferative erythron) Aspirin remains controversial Low-dose aspirin (for antiplatelet action) may be used if the platelet count remains high or thrombosis occurs despite the above treatment but is best avoided in patients with a history of haemorrhage Aplastic anaemia Marrow failure (aplastic anaemia) may be primary, of which 75% are idiopathic acquired, and 25% secondary to a variety of agents, including chemicals (e.g benzene), drugs and infections Treatment is chosen according to the severity of the cytopenia, the age of the patient, the availability of a suitable bone marrow donor and, less commonly, the cause (if known) Good supportive treatment is important The major therapeutic choice is between allogeneic bone marrow transplantation and immunesuppression, e.g with antilymphocytic globulin and ciclosporin; and perhaps haemopoietic growth factors (see above) Survival rates after allogeneic transplantation are in the region of 75-80% for data collected from transplant centres by the International Bone Marrow Transplant Registry, though chronic graft-versus-host disease causes continued morbidity Immunosuppression is used in patients who are not candidates for bone marrow transplantation due to age or to the lack of a donor (up to 70%) Horse antithymocyte globulin (ATG) or rabbit antilymphocyte globulin (ALG) induce haematological responses (transfusion-independence and freedom from infection) in 40-50% The addition of ciclosporin to ATG or ALG improves response rates to 70-80% and survival rates in responders to 90% Adverse effects of ATG and ALG include anaphylaxis, exacerbation of cytopenias and serum sickness Ciclosporin is nephrotoxic In refractory patients GCSF and erythropoetin can improve blood counts, as can androgens in some patients APLASTIC ANAEMIA GUIDETO FURTHER READING Andrews N C 1999 Disorders of iron metabolism New England Journal of Medicine 341:1986-1995 Botto L D, Moore C A, Khoury M J et al 1999 Neural tube defects New England Journal of Medicine 341:1509-1519 Castle W B 1966 Treatment of pernicious anaemia: historical aspects Clinical Pharmacology and Therapeutics 7: 347 Ferner R E et al 1989 Drugs in donated blood Lancet 2: 93-94 Oliveri N F 1999 The B-thalassemias New England Journal of Medicine 341: 99-109 Roy C N, Enns C A 2000 Iron homeostasis: new tales from the crypt Blood 96: 4020-027 Spivak J L 2000 The blood in systemic disorders Lancet 355:1707-1712 29 Steinberg M H 1999 Management of sickle cell disease New England Journal of Medicine 340:1021-1030 Stock W, Hoffman R 2000 White blood cells: nonmalignant disorders Lancet 355:1351-1357 Tefferi A 2000 Myelofibrosis with myeloid metaplasia New England Journal of Medicine 342:1255-1265 Toh B-H, van Driel I R, Gleeson P A1997 Pernicious anaemia New England Journal of Medicine 337: 1441-1448 Weatherall D }, Provan A B 2000 Red cells I: inherited anaemias Lancet 355:1169-1175 Weatherall D }, Provan A B 2000 Red cells II: acquired anaemias and polycythaemica Lancet 355:1260-1268 Young N S, Maciejewski J 1997 The pathophysiology of acquired aplastic anaemia New England Journal of Medicine 336:1365-1372 601 ... These interactions can be clinically important Ingestion should be separated by hours Ascorbic acid increases absorption (see above) but its use (200 mg/day) is not clinically important in routine... Medicine 341:1509-1519 Castle W B 1966 Treatment of pernicious anaemia: historical aspects Clinical Pharmacology and Therapeutics 7: 347 Ferner R E et al 1989 Drugs in donated blood Lancet 2:... enterohepatic circulation which can be interrupted by intestinal disease and hastens the onset of clinical deficiency), and is excreted via the kidney Body stores amount to about mg (mainly in the