Maturation Factors - Vit B12 and Folic Acid

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 short-lived 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 B 12 has some independent metabolic functions as well. The active coenzyme forms of B12 generated in the body are deoxyadenosylcobalamin (DAB12) and methylcobalamin (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.

This 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 B 12 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:

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

·      Glossitis, g.i. disturbances: damage to epithelial structures.

·  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

Cyanocobalamin: REDISOL, MACRABIN 35 μg/5 ml liq; 100, 500, 1000 μg inj.

Hydroxocobalamin: REDISOLH, MACRABINH 500, 1000 μg inj.

Methylcobalamin: BIOCOBAL, DIACOBAL, METHYLCOBAL 0.5 mg tab.

Methyl B₁₂ is the active coenzyme form of vit B12 for synthesis of methionine and Sadenosyl-methionine 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 lifelong. 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 + para-amino benzoic 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 anti-anaemia 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 carrier mediated 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. 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:

·      Inadequate dietary intake

·   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.

·      Biliary fistula; bile containing folate for recirculation is drained.

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

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

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

Manifestations Of Deficiency Are:

·       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.

·      Epithelial damage: glossitis, enteritis, diarrhoea, steatorrhoea.

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

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

Preparations And Dose

Folic acid: FOLVITE, FOLITAB 5 mg tab;

Liquid oral preparations and injectables are available only in combination formulation (see Tables 43.1 and 67.2). Oral therapy is adequate except when malabsorption is present or in severely ill patient—given i.m.

Dose: therapeutic 2 to 5 mg/day, prophylactic 0.5 mg/ day.

Folinic acid; CALCIUM LEUCOVORIN 3 mg/ml inj.

FASTOVORIN 3 mg, 15 mg amps, 50 mg vial; RECOVORIN 15 mg tab, 15 mg, 50 mg vial for inj.

Uses

1.  Megaloblastic Anaemias due to:

·       Nutritional folate deficiency; manifests earlier than vit B12 deficiency. Response occurs as quickly as with vit B12.

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

·               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.

·          Malabsorption syndromes: Tropical sprue, coeliac disease, idiopathic steatorrhoea, etc.

·       Antiepileptic therapy: Megaloblastic anaemia can occur due to prolonged phenytoin/ phenobarbitone therapy (see Ch. No. 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, 5formylTHFA) 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 Antianaemia 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.