Haematinics and Erythropoietin

Chapter 43

Haematinics and Erythropoietin

Haematinics These are substances required in the formation of blood, and are used for treatment of anaemias.

Anamia occurs when the balance between production and destruction of RBCs is disturbed by:

(a) Blood loss (acute or chronic) 

(b) Impaired red cell formation due to: 

Deficiency of essential factors, i.e. iron, vitamin B12, folic acid. 

Bone marrow depression (hypoplastic anaemia), erythropoietin deficiency.

(c) Increased destruction of RBCs (haemolytic anaemia)

In this chapter essential factors required for normal formation or pigmentation of RBCs will be covered.

IRON

Iron has for long been considered important for the body. Lauha bhasma (calcined iron) has been used in ancient Indian medicine. According to Greek thought Mars is the God of strength and iron is dedicated to Mars: thus, iron was used for weakness, which is common in anaemia. In 1713 iron was shown to be present in blood. In the early 19th century Blaud developed his famous ‘Blaud’s pill’ consisting of ferrous sulfate and potassium carbonate for anaemia. All important aspects of iron metabolism have been learned in the past 60 years.

Distribution of iron in body Iron is an essential body constituent. Total body iron in an adult is 2.5–5 g (average 3.5 g). It is more in men (50 mg/ kg) than in women (38 mg/kg). It is distributed into:

Haemoglobin (Hb) : 66% 

Iron stores as ferritin and hemosiderin: 25% 

Myoglobin (in muscles) : 3% 

Parenchymal iron (in enzymes, etc.) : 6% 

Haemoglobin is a protoporphyrin; each molecule having 4 iron containing haeme residues. It has 0.33% iron; thus loss of 100 ml of blood (containing 15 g Hb) means loss of 50 mg elemental iron. To raise the Hb level of blood by 1 g/dl— about 200 mg of iron is needed. Iron is stored only in ferric form, in combination with a large protein apoferritin.

Ferritin can get saturated to different extents; at full saturation it can hold 30% iron by weight. The most important storage sites are reticuloendothelial (RE) cells. Parenchymal iron occurs as prosthetic group in many cellular enzymes— cytochromes, peroxidases, catalases, xanthine oxidase and some mitochondrial enzymes. Though, the primary reflection of iron deficiency occurs in blood, severe deficiency affects practically every cell.

Daily requirement To make good average daily loss, iron requirements are:

Iron absorption

The average daily diet contains 10–20 mg of iron. Its absorption occurs all over the intestine, but majority in the upper part. Dietary iron is present either as haeme or as inorganic iron. Absorption of haeme iron is better (upto 35% compared to inorganic iron which averages 5%) and occurs directly without the aid of a carrier (Fig. 43.1).

However, it is a smaller fraction of dietary iron. The major part of dietary iron is inorganic and in the ferric form. It needs to be reduced to the ferrous form before absorption. Two separate iron transporters in the intestinal mucosal cells function to effect iron absorption. At the luminal membrane the divalent metal transporter 1 (DMT1) carrys ferrous iron into the mucosal cell. This along with the iron released from haeme is transported across the basolateral membrane by another iron transporter ferroportin (FP). These iron transporters are regulated according to the body needs. Absorption of haeme iron is largely independent of other foods simultaneously ingested, but that of inorganic iron is affected by several factors.

Factors facilitating iron absorption

1. Acid: by favouring dissolution and reduction of ferric iron. 

2. Reducing substances: ascorbic acid, amino acids containing SH radical. These agents reduce ferric iron and form absorbable complexes.

3. Meat: by increasing HCl secretion and providing haeme iron.

Factors impeding iron absorption

1. Alkalies (antacids) render iron insoluble, oppose its reduction. 

2. Phosphates (rich in egg yolk)

 3. Phytates (in maize, wheat) 

4. Tetracyclines 

5. Presence of other foods in the stomach. 

In general, bioavailability of iron from cereal based diets is low.

Mucosal block The gut has a mechanism to prevent entry of excess iron in the body. Iron reaching inside mucosal cell is either transported to plasma or oxidised to ferric form and complexed with apoferritin to form ferritin (Fig. 43.1). This ferritin generally remains stored in the mucosal cells and is lost when they are shed (lifespan 2–4 days). This is called the ‘Ferritin curtain’. The iron status of the body and erythropoietic activity govern the balance between these two processes, probably through a ‘haematopoietic transcription factor’, and thus the amount of iron that will enter the body. A larger percentage is absorbed during iron deficiency. When body iron is low or erythropoiesis is occurring briskly, ferritin is either not formed or dissociates soon— the released iron is transported to the blood. Mucosal block however, can be overwhelmed by gross excess of iron.

Transport, utilization, storage and excretion

Free iron is highly toxic. As such, on entering plasma it is immediately converted to the ferric form and complexed with a glycoprotein transferrin (Tf). Iron circulates in plasma bound to Tf (two Fe3+ residues per molecule). The total plasma iron content (~3 mg) is recycled 10 times everyday (turnover of iron is 30 mg/day).

Iron is transported into erythropoietic and other cells through attachment of transferrin to specific membrane bound transferrin receptors (TfRs). The complex is engulfed by receptor mediated endocytosis. Iron dissociates from the complex at the acidic pH of the intracellular vesicles; the released iron is utilized for haemoglobin synthesis or other purposes, while Tf and TfR are returned to the cell surface to carry fresh loads. In iron deficiency and haemolytic states when brisk erythropoiesis is occurring, TfRs in erythropoietic cells increase in number. This does not occur in other cells. Thus, the erythron becomes selectively more efficient in trapping iron.

Iron is stored in RE cells in liver, spleen, bone marrow, also in hepatocytes and myocytes as ferritin and haemosiderin after entering these cells through TfRs. Apoferritin synthesis is regulated by iron status of the body. When it is low—the ‘iron regulating element’ (IRE) on mRNA is blocked—transcription of apoferritin does not occur, while more Tf is produced. On the other hand, more apoferritin is synthesized to trap iron when iron stores are rich. Plasma iron derived from destruction of old RBCs (lifespan ~120 days), from stores and from intestinal absorption forms a common pool that is available for erythropoiesis, to all other cells and for restorage.

Iron is tenaciously conserved by the body; daily excretion in adult male is 0.5–1 mg, mainly as exfoliated g.i. mucosal cells, some RBCs and in bile (all lost in faeces). Other routes are desquamated skin, very little in urine and sweat. In menstruating women, monthly menstrual loss may be averaged to 0.5–1 mg/day. Excess iron is required during pregnancy for expansion of RBC mass, transfer to foetus and loss during delivery; totalling to about 700 mg. This is to be met in the later 2 trimesters.

Preparations and dose

Oral iron

The preferred route of iron administration is oral. Dissociable ferrous salts are inexpensive, have high iron content and are better absorbed than ferric salts, especially at higher doses. Gastric irritation and constipation (the most important side effects of oral iron) are related to the total quantity of elemental iron administered. If viewed in terms of iron content, nearly all preparations have the same degree of gastric tolerance, the

limits of which are fairly well defined in individual patients. Some simple oral preparations are ;

1. Ferrous sulfate: (hydrated salt 20% iron, dried salt 32% iron) is the cheapest; may be preferred on this account. It often leaves a metallic taste in mouth;

FERSOLATE 200 mg tab.

2. Ferrous gluconate (12% iron):

FERRONICUM 300 mg tab, 400 mg/15 ml elixer.

3. Ferrous fumarate (33% iron): is less water soluble than ferrous sulfate and tasteless;

NORI-A 200 mg tab.

4. Colloidal ferric hydroxide (50% iron): 

NEOFERUM 200 mg tab, 400 mg/5 ml liquid, 100 mg/ml drops.

Other forms of iron present in oral formulations are:

Ferrous succinate (35% iron) Iron choline citrate Iron calcium complex (5% iron) Ferric ammonium citrate (scale iron) Ferrous aminoate (10% iron) Ferric glycerophosphate Iron hydroxy polymaltose .

These are claimed to be better absorbed and/or produce less bowel upset, but this is primarily due to lower iron content. They are generally more expensive.

A number of oral formulations containing one of the iron compounds along with one to many vitamins, yeast, amino acids and other minerals are widely marketed and promoted. Some of these are listed in Table 43.1, but should be considered irrational.

A technical Advisory Board (India) has recommended that B complex vitamins and zinc should not be included in iron and folic acid containing haematinic preparations. Iron hydroxy polymaltose has been marketed by many pharmaceuticals and vigorously promoted for its high iron content, no metallic taste, good g.i. tolerability and direct absorption from the intestines. Because the complex releases little free iron in the gut lumen—g.i. irritation is minimal. However, the high bioavailability observed in rats has not been found in humans and reports of its poor efficacy in treating iron deficiency anaemia have appeared. Preparations of iron hydroxy polymaltose are 4–5 times costlier than other iron salts and its therapeutic efficacy is questionable.

The elemental iron content and not the quantity of iron compound per dose unit should be taken into consideration. Sustained release preparations are more expensive and not rational because most of the iron is absorbed in the upper intestine, while these preparations release part of their iron content lower down. Liquid formulations may stain teeth: should be put on the back of tongue and swallowed. In general they are less satisfactory.

A total of 200 mg elemental iron (infants and children 3–5 mg/kg) given daily in 3 divided doses produces the maximal haemopoietic response. Prophylactic dose is 30 mg iron daily. Absorption is much better when iron preparations are taken in empty stomach. However, side effects are also more; some prefer giving larger amounts after meals, while others like to give smaller doses in between meals.

Adverse effects of oral iron These are common at therapeutic doses and are related to elemental iron content. Individuals differ in susceptibility. Epigastric pain, heartburn, nausea, vomiting, staining of teeth, metallic taste, bloting, colic.

Constipation is more common (believed to be due to astringent action of iron) than diarrhoea (thought to reflect irritant action). However, these may be caused by alteration of intestinal flora as well.

Parenteral iron

Iron therapy by injection is indicated only when:

1. Oral iron is not tolerated: bowel upset is too much. 

2. Failure to absorb oral iron: malabsorption; inflammatory bowel disease. Chronic inflammation (rheumatoid arthritis) decreases iron absorption, also the rate at which iron can be utilized is decreased.

 3. Non-compliance to oral iron. 

4. In presence of severe deficiency with chronic bleeding. 

5. Along with erythropoietin: oral ion may not be absorbed at sufficient rate to meet the demands of induced rapid erythropoiesis. Parenteral iron therapy needs calculation of the total iron requirement of the patient.

Iron requirement (mg) = 4.4 × body weight (kg) × Hb deficit (g/dl)

This formula includes iron needed for replenishment of stores. The rate of response with parenteral iron is not faster than with optimal doses given orally. However, stores can be replenished in a shorter time by parenteral therapy.

The ionized salts of iron used orally, cannot be injected because of their strong protein precipitating action. Two organically complexed preparations for parenteral use are:

(i) Iron-dextran: as a colloidal solution containing 50 mg elemental iron/ml is the preparation of choice;

 IMFERON 2 ml ampoule

(ii) Iron-sorbitol-citric acid complex: 50 mg iron/ml;

 JECTOFER 1.5 ml ampoule. 

The i.m. dose of both iron-dextran and ironsorbitol is 30% higher than the calculated requirement of a patient. A test dose of the preparation (few drops) must be injected first to screen sensitive patients. 

Intramuscular: Injection is given deeply in the gluteal region using Z track technique (to avoid staining of the skin). Iron dextran can be injected 2 ml daily, or on alternate days, or 5 ml each side on the same day (local pain lasting weeks may occur with the higher dose). More than 1.5–2 ml of iron-sorbitol should not be injected at one time.

Intravenous: After a test dose of 0.5 ml iron-dextran injected i.v. over 5–10 min, 2 ml can be injected per day taking 10 min for the injection. Alternatively the total calculated dose is diluted in 500 ml of glucose/saline solution and infused i.v. over 6–8 hours under constant observation. Injection should be terminated if the patient complains of giddiness, paresthesias or constriction in chest. Intravenous iron injection is more risky than i.m. injection. Iron sorbitol is not suitable for i.v. use or for total dose infusion because it would rapidly saturate transferrin and very high levels of free iron in blood will be attained.

Adverse effects of parenteral iron

Local Pain at site of i.m. injection, pigmentation of skin, sterile abscess—especially in old and debilitated patient.

Systemic Fever, headache, joint pains, flushing, palpitation, chest pain, dyspnoea, lymph node enlargement. A metallic taste in mouth lasting few hours occurs with iron-sorbitol injection. An anaphylactoid reaction resulting in vascular collapse and death occurs rarely. Iron sorbitol causes more immediate reactions than irondextran. Iron-sorbitol should be avoided in patients with kidney disease.

Use

1. Iron deficiency anaemia It is the most important indication for medicinal iron. Iron deficiency is the commonest cause of anaemia, especially in developing countries where a sizable percentage of population is anaemic. The RBC are microcytic and hypochromic due to deficient Hb synthesis. Other metabolic manifestations are seen when iron deficiency is severe. Apart from nutritional deficiency, chronic bleeding from g.i. tract (ulcers, hookworm infestation) is a common cause. Iron deficiency also accompanies repeated attacks of malaria and chronic inflammatory diseases. The cause of iron deficiency should be identified and treated. Iron should be normally administered orally; parenteral therapy is to be reserved for special circumstances. A rise in Hb level by 0.5– 1 g/dl per week is an optimum response to iron therapy. It is faster in the beginning and when anaemia is severe. Later, the rate of increase in Hb% declines. However, therapy should be continued till normal Hb level is attained (generally takes 1–3 months depending on the severity) and 2–3 months thereafter to replenish the stores, because after correction of anaemia, iron absorption is slow.

Prophylaxis: The amount of iron available from average diet and the absorptive processes in the intestine place a ceiling on iron absorption of ~3 mg/day. Thus, iron balance is precarious in most menstruating women. Later half of pregnancy and infancy are periods when iron deficiency will develop unless medicinal iron is supplemented.

In these situations as well as others (chronic illness, menorrhagia, after acute blood loss, etc.) prophylactic use of iron is indicated.

2. Megaloblastic anaemia When brisk haemopoiesis is induced by vit B12 or folate therapy, iron deficiency may be unmasked. The iron status of these patients should be evaluated and iron given accordingly.

3. As an astringent Ferric chloride is used in throat paint.

ACUTE IRON POISONING

It occurs mostly in infants and children: 10–20 iron tablets or equivalent of the liquid preparation (> 60 mg/kg iron) may cause serious toxicity in them. It is very rare in adults. Manifestations are vomiting, abdominal pain, haematemesis, diarrhoea, lethargy, cyanosis, dehydration, acidosis, convulsions; finally shock, cardiovascular collapse and death. In few cases death occurs early (within 6 hours), but is typically delayed to 12– 36 hours, with apparent improvement in the intervening period. The pathological lesion is haemorrhage and inflammation in the gut, hepatic necrosis and brain damage.

Treatment It should be prompt.

To prevent further absorption of iron from gut

a) Induce vomiting or perform gastric lavage with sodium bicarbonate solution—to render iron insoluble.

(b) Give egg yolk and milk orally: to complex iron. Activated charcoal does not adsorb iron.

To bind and remove iron already absorbed

Desferrioxamine (an iron chelating agent—see Ch. 66) is the drug of choice. It should be injected i.m. (preferably) 0.5–1 g (50 mg/kg) repeated 4–12 hourly as required, or i.v. (if shock is present) 10–15 mg/kg/hour; max 75 mg/kg in a day till serum iron falls below 300 μg/dl. Early therapy with desferrioxamine has drastically reduced mortality of iron poisoning.

Alternatively DTPA or calcium edetate (see Ch. 66) may be used if desferrioxamine is not available. BAL is contraindicated because its iron chelate is also toxic.

Supportive measures Fluid and electrolyte balance should be maintained and acidosis corrected by appropriate i.v. infusion. Respiration and BP may need support. Diazepam i.v. should be cautiously used to control convulsions, if they occur.

Miscellaneous/Adjuvant haematinics

1. Copper Haeme synthesis is interfered in copper deficiency. However, copper is a trace metal for man and clinical deficiency is very rare. Its routine use is, therefore, not justified. However, when copper deficiency is demonstrated, 0.5–5 mg of copper sulphate/day may be given therapeutically; prophylactic dose is 0.1 mg/day. It is present in some haematinic combinations (see Table 43.1).

2. Cobalt It stimulates erythropoiesis transiently, probably by inducing tissue hypoxia → increased erythropoietin production. Cobalt deficiency is not known in man. Moreover, it can cause hypothyroidism, angina and CHF. It should not be prescribed.

3. Pyridoxine (see Ch. 67) Pyridoxine responsive anaemia is a rare entity. It is due to inherent abnormality in haeme synthesis. Sideroblastic anaemia associated with isoniazid and pyrazinamide (which interfere with pyridoxine metabolism and action) therapy needs to be treated with pyridoxine. Some other sideroblastic anaemias show partial improvement with large doses of pyridoxine. However, routine use of pyridoxine in anaemia is wasteful.

4. Riboflavin (see Ch. 67) Hypoplastic anaemia occurs in riboflavin deficiency which is generally a part of multiple deficiencies in protein-calorie malnutrition. In the absence of specific deficiency, use of riboflavin in anaemia is of no value.

MATURATION FACTORS

Deficiency of vit B12 and folic acid, which are B group vitamins, results in megaloblastic anaemia characterized by the presence of large red cell precursors in bone marrow and their large and shortlived progeny in peripheral blood. Vit B12 and folic acid are therefore called maturation factors. The basic defect is in DNA synthesis. Apart from haemopoietic, other rapidly proliferating tissues also suffer.

VITAMIN-B12

Cyanocobalamin and hydroxocobalamin are complex cobalt containing compounds present in the diet and referred to as vit B12.

Thomas Addison (1849) described cases of anaemia not responding to iron. This was later called ‘pernicious’ (incurable, deadly) anaemia and its relation with atrophy of gastric mucosa was realized. Minot and Murphy (1926) treated such patients by including liver in diet and received Nobel prize. Castle (1927–32) propounded the hypothesis that there was an extrinsic factor present in diet which combined with an intrinsic factor produced by stomach to give rise to the haemopoietic principle. Vit B12 was isolated in 1948 and was shown to be the extrinsic factor as well as the haemopoietic principle, the intrinsic factor only helped in its absorption.

Vit B12 occurs as water soluble, thermostable red crystals. It is synthesized in nature only by microorganisms; plants and animals acquire it from them.

Dietary sources Liver, kidney, sea fish, egg yolk, meat, cheese are the main vit B12 containing constituents of diet. The only vegetable source is legumes (pulses) which get it from microorganisms harboured in their root nodules. Vit B12 is synthesized by the colonic microflora but this is not available for absorption in man. The commercial source is Streptomyces griseus; as a byproduct of streptomycin industry.

Daily requirement: 1–3 μg, pregnancy and lactation 3–5 μg.

Metabolic functions Vit B12 is intricately linked with folate metabolism in many ways; megaloblastic anaemia occurring due to deficiency of either is indistinguishable. In addition, vit B12 has some independent metabolic functions as well. The active coenzyme forms of B12 generated in the body are deoxyadenosyl-cobalamin (DAB12) and methyl-cobalamin (methyl B12).

(i) Vit B12 is essential for the conversion of homocysteine to methionine.

Methionine is needed as a methyl group donor in many metabolic reactions and for protein synthesis. This reaction is also critical in making tetrahydrofolic acid (THFA) available for reutilization. In B12 deficiency THFA gets trapped in the methyl form and a number of one carbon transfer reactions suffer (see under folic acid). 

(ii) Purine and pyrimidine synthesis is affected primarily due to defective ‘one carbon’ transfer because of ‘folate trap’. The most important of these is inavailability of thymidylate for DNA production.

is an important step in propionic acid metabolism. It links the carbohydrate and lipid metabolisms. This reaction does not require folate and has been considered to be responsible for demyelination seen in B12 deficiency, but not in pure folate deficiency. That myelin is lipoidal, supports this contention.

(iv)Now it appears that interference with the reaction:

may be more important in the neurological damage of B12 deficiency, because it is needed in the synthesis of phospholipids and myelin.

(v) Vit B12 is essential for cell growth and multiplication.

Utilization of vit B12

Vit B12 is present in food as protein conjugates and is released by cooking or by proteolysis in stomach facilitated by gastric acid. Intrinsic factor (a glycoprotein, MW60,000) secreted by stomach forms a complex with B12— attaches to specific receptors present on intestinal mucosal cells and is absorbed by active carrier mediated transport. This mechanism is essential for absorption of vit B12 ingested in physiological amounts. However, when gross excess is taken, a small fraction is absorbed without the help of intrinsic factor.

Vit B12 is transported in blood in combination with a specific β globulin transcobalamin II (TCII). Congenital absence of TCII or presence of abnormal protein (TCI or TCIII, in liver and bone marrow disease) may interfere with delivery of  vit B12 to tissues. Vit B12 is especially taken up by liver cells and stored: about 2/3 to 4/5 of body’s content (2–8 mg) is present in liver.

Vit B12 is not degraded in the body. It is excreted mainly in bile (3–7 μg/day); all but 0.5– 1 μg of this is reabsorbed—considerable enterohepatic circulation occurs. Thus, in the absence of intrinsic factor or when there is malabsorption, B12 deficiency develops much more rapidly than when it is due to nutritional deficiency. It takes 3–5 years of total absence of B12 in diet to deplete normal body stores.

Vit B12 is directly and completely absorbed after i.m. or deep s.c. injection. Normally, only traces of B12 are excreted in urine, but when pharmacological doses (> 100 μg) are given orally or parenterally—a large part is excreted in urine, because the plasma protein binding sites get saturated and free vit B12 is filtered at the glomerulus. Hydroxocobalamin is more protein bound and better retained than cyanocobalamin.

Deficiency Vit B12 deficiency occurs due to:

1. Addisonian pernicious anaemia: is probably an autoimmune disorder which results in destruction of gastric parietal cells → absence of intrinsic factor in gastric juice (along with achlorhydria) → inability to absorb vit B12. 

2. Other causes of gastric mucosal damage, e.g. chronic gastritis, gastric carcinoma, gastrectomy, etc.

3. Malabsorption (damaged intestinal mucosa), bowel resection. 

4. Consumption of vit B12 by abnormal flora in intestine (blind loop syndrome) or fish tape worm.

5. Nutritional deficiency: less common cause. 

6. Increased demand: pregnancy, infancy.

Manifestations of deficiency are:

(a) Megaloblastic anaemia (generally the first manifestation), neutrophils with hypersegmented nuclei, giant platelets. 

(b) Glossitis, g.i. disturbances: damage to epithelial structures. 

(c) Neurological: subacute combined degeneration of spinal cord; peripheral neuritis—diminished vibration and position sense, paresthesias, depressed stretch reflexes; mental changes—poor memory, mood changes, hallucinations, etc. are late effects.

Preparations, dose, administration

Methyl B12 is the active coenzyme form of vit B12 for synthesis of methionine and S-adenosylmethionine that is needed for integrity of myelin. This preparation of vit B12 in a dose of 1.5 mg/ day has been especially promoted for correcting the neurological defects in diabetic, alcoholic and other forms of peripheral neuropathy. However, in USA and many other countries, it is used only as a nutritional supplement, and not as a drug.

Combination preparations of B12 with other vitamins and iron are listed in Tables 43.1 and 67.2. Hydroxocobalamin has been preferred for parenteral use because of better retention. However, it has been found to induce antibody formation so that vit B12 becomes metabolically unavailable. It is not recommended in USA, but used in UK and India.

When vit B12 deficiency is due to lack of intrinsic factor (pernicious anaemia and other causes), it should be given by i.m. or deep s.c. (but not i.v.) injection. Parenteral administration is necessary to bypass the defective absorptive mechanism. Initially 30–100 μg/day for 10 days followed by 100 μg weekly and then monthly for maintenance—indefinitely or life-long. When neurological complications are present, a higher dose (500–1000 μg/day) has been used, but the response is not superior to conventional doses.

In other types of deficiency 10–30 μg/day may be used orally. The prophylactic dose is 3–10 μg/ day.

Uses

1. Treatment of vit B12 deficiency: vit B12 is used as outlined above. It is wise to add 1–5 mg of oral folic acid and an iron preparation, because reinstitution of brisk haemopoiesis may unmask deficiency of these factors. Response to vit B12 is dramatic—symptomatic improvement starts in 2 days: appetite improves, patient feels better; mucosal lesions heal in 1–2 weeks; reticulocyte count increases; Hb% and haematocrit rise progressively; platelet count normalises in 10 days and WBC count in 2–3 weeks. Time taken for complete recovery of anaemia depends on the severity of disease to start with. Neurological parameters improve more slowly—may take several months; full recovery may not occur if vit B12 deficiency has been severe or had persisted for long.

2. Prophylaxis: needs to be given only when there are definite predisposing factors for development of deficiency (see above).

3. Mega doses of vit B12 have been used in neuropathies, psychiatric disorders, cutaneous sarcoid and as a general tonic to allay fatigue, improve growth—value is questionable. 4. Tobacco amblyopia: hydroxocobalamin is of some benefit—it probably traps cyanide derived from tobacco to form cyanocobalamin.

Adverse effects Even large doses of vit B12 are quite safe. Allergic reactions have occurred on injection, probably due to contaminants. Anaphylactoid reactions (probably to sulfite contained in the formulation) have occurred on i.v. injection: this route should not be employed.

FOLIC ACID

It occurs as yellow crystals which are insoluble in water, but its sodium salt is freely water soluble. Chemically it is Pteroyl glutamic acid (PGA) consisting of pteridine + paraaminobenzoic acid (PABA) + glutamic acid.

Wills (1932–37) had found that liver extract contained a factor, other than vit B12, which could cure megaloblastic anaemia. Mitchell in 1941 isolated an antianaemia principle from spinach and called it ‘folic acid’ (from leaf). Later the Will’s factor was shown to be identical to folic acid.

Dietary sources Liver, green leafy vegetables (spinach), egg, meat, milk. It is synthesized by gut flora, but this is largely unavailable for absorption.

Daily requirement of an adult is < 0.1 mg but dietary allowance of 0.2 mg/day is recommended. During pregnancy, lactation or any condition of high metabolic activity, 0.8 mg/ day is considered appropriate.

Utilization Folic acid is present in food as polyglutamates; the additional glutamate residues are split off primarily in the upper intestine before being absorbed. Reduction to DHFA and methylation also occurs at this site. It is transported in blood mostly as methyl-THFA which is partly bound to plasma proteins. Small, physiological amounts of folate are absorbed by specific carriermediated active transport in the intestinal mucosa. Large pharmacological doses may gain entry by passive diffusion, but only a fraction is absorbed.

Folic acid is rapidly extracted by tissues and stored in cells as polyglutamates. Liver takes up a large part and secretes methyl-THFA in bile which is mostly reabsorbed from intestine: enterohepatic circulation occurs. Alcohol interferes with release of methyl-THFA from hepatocytes. The total body store of folates is 5–10 mg. Normally, only traces are excreted, but when pharmacological doses are given, 50–90% of a dose may be excreted in urine.

Metabolic functions Folic acid is inactive as such and is reduced to the coenzyme form in two steps: FA → DHFA → THFA by folate reductase (FRase) and dihydrofolate reductase (DHFRase). THFA mediates a number of one carbon transfer reactions by carrying a methyl group as an adduct (see under vit. B12 also).

1. Conversion of homocysteine to methionine: vit B12 acts as an intermediary carrier of methyl group (see p. 588). This is the most important reaction which releases THFA from the methylated form.

2. Generation of thymidylate, an essential constituent of DNA:

3. Conversion of serine to glycine: needs THFA and results in the formation of methylene-THFA which is utilized in thymidylate synthesis.

4. Purine synthesis: de novo building of purine ring requires formyl-THFA and methenyl-THFA (generated from methylene-THFA) to introduce carbon units at position 2 and 8

5. Generation and utilization of ‘formate pool’.

6. Histidine metabolism: for mediating formimino group transfer.

Ascorbic acid protects folates in the reduced form. Other cofactors, e.g. pyridoxal, etc. are required for some of the above reactions.

Deficiency Folate deficiency occurs due to:

(a) Inadequate dietary intake

 (b) Malabsorption: especially involving upper intestine— coeliac disease, tropical sprue, regional ileitis, etc. Deficiency develops more rapidly as both dietary and biliary folate is not absorbed.

(c) Biliary fistula; bile containing folate for recirculation is drained. 

(d) Chronic alcoholism: intake of folate is generally poor. Moreover, its release from liver cells and recirculation are interfered.

(e) Increased demand: pregnancy, lactation, rapid growth periods, haemolytic anaemia and other diseases with high cell turnover rates. 

(f) Drug induced: prolonged therapy with anticonvulsants (phenytoin, phenobarbitone, primidone) and oral contraceptives—interfere with absorption and storage of folate.

Manifestations of deficiency are:

(i) Megaloblastic anaemia, indistinguishable from that due to vit B12 deficiency. However, folate deficiency develops more rapidly if external supply is cut off: body stores last 3–4 months only. In malabsorptive conditions megaloblastosis may appear in weeks.

(ii) Epithelial damage: glossitis, enteritis, diarrhoea, steatorrhoea.

(iii) Neural tube defects, including spina bifida in the offspring, due to maternal folate deficiency. 

(iv) General debility, weight loss, sterility. However, neurological symptoms do not appear in pure folate deficiency.

Preparations and dose

Uses

1. Megaloblastic anaemias due to:

(a) Nutritional folate deficiency; manifests earlier than vit B12 deficiency. Response occurs as quickly as with vit B12.

(b) Increased demand: pregnancy, lactation, infancy, during treatment of severe iron deficiency anaemia, haemolytic anaemias.

(c) Pernicious anaemia: folate stores may be low and deficiency may be unmasked when vit B12 induces brisk haemopoiesis: it has only secondary and adjuvant role in this condition.

Folic acid should never be given alone to patients with vit B12 deficiency—haematological response may occur, but neurological defect may progress due to diversion of meagre amount of vit B12 present in body to haemopoiesis.

(d) Malabsorption syndromes: Tropical sprue, coeliac disease, idiopathic steatorrhoea, etc.

(e) Antiepileptic therapy: Megaloblastic anaemia can occur due to prolonged phenytoin/ phenobarbitone therapy (see Ch. 30). This is treated by folic acid, but large doses should be avoided as they may antagonize anticonvulsant effect.

2. Prophylaxis of folate deficiency: only when definite predisposing factors are present. Routine folate supplementation (1 mg/day) is recommended during pregnancy to reduce the risk of neural tube defects in the newborn.

3. Methotrexate toxicity Folinic acid (Leucovorin, citrovorum factor, 5-formyl-THFA) is an active coenzyme form which does not need to be reduced by DHFRase before it can act. Methotrexate is a DHFRase inhibitor; its toxicity is not counteracted by folic acid, but antagonized by folinic acid.

Folinic acid is expensive and not needed for the correction of simple folate deficiency for which folic acid is good enough.

4. Citrovorum factor rescue In certain malignancies, high dose of methotrexate is injected i.v. and is followed within ½–1 hour with 1–3 mg i.v. of folinic acid to rescue the normal cells. It is ineffective if given > 3 hours after methotrexate.

Adverse effects Oral folic acid is entirely nontoxic. Injections rarely cause sensitivity reactions.

Shotgun antianemia preparations A large number of formulations containing varying quantities of iron, vit B12, folic acid and may be other vitamins and nutrients are marketed and promoted. They are liable to be used indiscriminately without proper assessment of needs of the patient, and investigating the cause of anaemia. Most preparations contain one or all ingredients in low amounts; thus, an incomplete response can occur. Diagnosis and assessment of the patient can become impossible thereafter.

ERYTHROPOIETIN

Erythropoietin (EPO) is a sialoglycoprotein hormone (MW 34000) produced by peritubular cells of the kidney. It is essential for normal erythropoiesis. Anaemia and hypoxia are sensed by kidney cells → rapid secretion of EPO → acts on erythroid marrow and:

a) Stimulates proliferation of colony forming cells of the erythroid series.

(b) Induces haemoglobin formation and erythroblast maturation.

(c) Releases reticulocytes in circulation.

EPO binds to specific receptors on the surface of its target cells. The EPO receptor is a JAK-STATkinase binding receptor that alters phosphorylation of intracellular proteins and activates transcription factors to regulate gene expression. It induces erythropoiesis in a dose dependent manner, but has no effect on RBC lifespan.

 The recombinant human erythropoietin (Epoetin α, β) is administered by i.v. or s.c. injection and has a plasma t½ of 6–10 hr.

Use The primary indication for epoetin is anaemia of chronic renal failure which is due to low levels of EPO; 25–100 U/kg s.c. or i.v. 3 times a week (max. 600 U/kg/week) raises haematocrit and haemoglobin, reduces need for transfusions and improves quality of life. It is prudent to start with a low dose and titrate upwards to keep haematocrit between 30–36%, and Hb 10–12 g/ dl. Some recent studies have indicated that dose reduction by about 30% is possible when epoetin is given s.c. compared to i.v. Exercise capacity and overall wellbeing of the patients is improved. Most patients have low iron stores; require concurrent parenteral/oral iron therapy for an optimum response. Other uses are:

1. Anaemia in AIDS patients treated with zidovudine. 

2. Cancer chemotherapy induced anaemia. 

3. Preoperative increased blood production for autologous transfusion during surgery.

Adverse effects Epoetin is nonimmunogenic. Adverse effects are related to sudden increase in haematocrit, blood viscosity and peripheral vascular resistance (due to correction of anaemia). These are—increased clot formation in the A-V shunts (most patients are on dialysis) hypertensive episodes, occasionally seizures. Flu like symptoms lasting 2–4 hr occur in some patients.

HEMAX 2000 IU/ml and 4000 IU/ml vials; EPREX 2000 IU, 4000 IU and 10000 IU in 1 ml prefilled syringes; ZYROP (epoetin β) 2000 IU and 4000 IU vials.