Introduction

A major distinction in diagnosis and classification of anemias is whether the eventual red cells that appear in the circulation are smaller (microcytic) or larger (macrocytic) than the usual normal cell size (normocytic). The most important example of the former is iron deficiency anemia where it appears that the red cell precursors, during their replication in the bone marrow from an original pluripotent stem cell undergo a higher than normal number of divisions. Since each such division results in two daughter cells that are slightly smaller, an increase in the number of divisions in the marrow compartment will result in smaller red cells in the circulation. In iron deficiency this is thought to happen because the usual progressive inactivation of the nucleus after each division occurs at a slower than normal rate.

The most characteristic example of a macrocytic anemia occurs because there is an abnormally slow rate of DNA biosynthesis in the developing red cell. Such reduced synthesis delays the rate of development of the nucleus and with it the rate of cell division during replication in the bone marrow compartment. Thus, by the time such cells have differentiated to the point at which they receive a signal to leave the bone marrow, they have undergone fewer than usual cell divisions, resulting in cells that are larger than normal

Pyruvate

Pyruvate

Figure 1 The role of folate cofactors in the DNA and methylation cycles.

or macrocytic. The other unique characteristic of such arrest in DNA biosynthesis is evidenced by the cells that are present in the bone marrow itself. The red cell precursors have a very different appearance from that of the normally developing red cell series (normoblasts). The nuclei are much larger than usual and are far less differentiated. These characteristic cells are called megaloblasts and only occur where there has been a slow down or arrest of DNA biosynthesis. This occurs in only three circumstances: folate deficiency, vitamin B12 deficiency, or during therapy with drugs that interfere directly or indirectly with DNA biosynthesis (Figure 1).

Definition

As the name suggests and as discussed above the unique feature that defines megaloblastic anemia is the presence of abnormal red cell precursors called megaloblasts in the bone marrow. Therefore, bone marrow examination by a competent hematologist remains the gold standard for diagnosis of megalo-blastic anemia. As discussed below, such morphological examinations are no longer routinely part of the diagnosis. However, despite the availability of other tests, the presence of megaloblasts in bone marrow aspirate remains the only way to achieve a definitive diagnosis and is still required if the patient fails to respond to treatment.

Biochemical Aspects of the Megaloblastic Anemias

The biochemical interrelationships between vitamin B12 and folate are described in Figure 1 and discussed elsewhere in the chapters on cobalamins and folic acid. The folate cofactors are essential for the provision of so-called carbon one units for the biosynthesis of purines and pyrimidines and thus for DNA. Folate in the form of 5-methyltetrahydrofo-late (5-methyl THF) is also needed to supply the methylation cycle with methyl groups (Figure 1). These are needed to regenerate methionine and S-adenosylmethionine (SAM) in cells, the latter being used to donate methyl groups to the three dozen or so methyltransferases present in all cells. In hepatocytes, part of the methylation cycle is used to degrade the 60% or so excess of methionine present in the diet over and above daily requirements. When folate status is reduced there will be a reduced capacity in cells to make DNA and thus to replicate. This will be most easily seen in rapidly dividing cells such as those of the bone marrow, hence the emergence of the very characteristic megaloblastic anemia with megaloblasts being seen in bone marrow aspirates. Clearly one would also expect to see a reduction in the methylation cycle, which could in turn reduce the activity of the numerous methyltransferases. The effects of such a reduction are less obvious and contrast sharply with what happens when the methylation cycle is interrupted by deficiency of vitamin B12 (see below). Vitamin B12 is involved in two enzymatic reactions in man, methylmalonyl CoA mutase and methionine synthase. As discussed later deficiency of the former leads to a raised level of methylmalonyl CoA in cells, which is seen in the circulation and the urine as methylmalonic acid (MMA). What is of very great interest is the clinical sequence of events during vitamin B12 deficiency and how they arise. There are two such sequences: the development of a megaloblastic anemia identical to that seen in folate deficiency and a neuropathy not usually associated with folate deficiency.

At a biochemical level, the explanation for the anemia is encapsulated in the methyl trap hypothesis, first put forward by Victor Herbert as early as 1961. The biosynthesis of 5-methyl THF by the enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) (Figure 1) is irreversible in vivo. Thus, once formed this folate cofactor can only be used by the vitamin B12-dependent enzyme methionine synthase. The activity of the enzyme is reduced or absent in the bone marrow of patients with vitamin B12 deficiency. Progressively more and more of the folate cofactors become metabolically trapped as 5-methyl THF reducing the intracellular levels of 10-formyl THF and 5,10-methylene THF needed for the biosynthesis of purines and pyrimidines and thus for DNA and cell division. Although such cells contain folate, they are unable to use it and suffer from a kind of pseudo folate deficiency, thus producing an identical megaloblastic anemia to that seen in folate deficiency. One might question why cells do such an apparently destructive thing. The answer is that cells perceive vitamin B12 deficiency through an ever-reducing level of SAM. This essential methyl donor normally downregulates the amount of 5-methyl THF synthesized in cells by reducing the level of the enzyme MTHFR. Falling levels of SAM in vitamin B12 deficiency by contrast are met with an ever-increasing activity of MTHFR and diversion of the folate cofactors into the trapped form, namely 5-methyl THF, which, of course, cannot be regenerated into THF because of the absence of methionine synthase.

A very characteristic neuropathy is also associated with vitamin B12 deficiency. This neuropathy is due to a reduction or interruption of the methylation cycle. This is clear from two independent lines of evidence. Firstly, inactivation of methionine synthase in experimental animals (monkeys and pigs) leads to the classical so-called subacute combined degeneration of the spinal cord (SCD) seen in patients with severe vitamin B12 deficiency. Secondly, patients with genetically very rare dramatic reductions in the enzyme MTHFR have the classical signs and symptoms of SCD. The most plausible explanation is that as a result of reduced MTHFR levels they are unable to supply the methyl groups needed for the methylation cycle. It is of interest that such patients do not get megaloblastic anemia, presumably because while their folate metabolism is interfered with, there is no trapping of the folate cofactors metabolically as 5-methyl THF. It seems probable that a reduction in activity of one or more of the methyltransferases, the activity of which is compromised by an interruption of the methylation cycle, causes the characteristic neuropathy. It is unclear which specific methyltransferase is involved.

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