José Luis Fedele

Megaloblastic anemias are characterized by a decrease in hemoglobin levels, accompanied by the presence in the bone marrow of megaloblasts, which are morphologically altered erythroblasts as a consequence of a maturation modification caused, most of the time, by the deficiency of some of the essential elements for the maturation of the nucleus (DNA synthesis), that is, vitamin B12 and folic acid.

These changes basically consist of an increase in cell size, with an immature nucleus and a cytoplasm with almost normal hemoglobin content, which is also known as alteration of nucleus-cytoplasmic maturation.

The deficiency of folic and B12 not only affects the red series, but also the entire hematopoiesis as a whole and other tissues of the body with intense regenerative activity (epithelia and mucous membranes).

Similarly, nervous tissue is affected by the deficiency of one of these essential vitamin elements, vitamin B12 (but not folic acid), which causes, specifically at this level, demyelination of the lateral and posterior cords of the spinal cord, a phenomenon known as combined latero-posterior degeneration.

Returning to the hemopoietic system, the deficits of B12 and folic acid are so characteristic that the therapeutic response to these elements in the case of megaloblastic anemia constitutes in itself a diagnostic criterion.

The pathophysiological mechanism of megaloblastic anemia is twofold.

On the one hand, ineffective erythropoiesis is generated, that is, destruction of the medullary elements in the maturing process before they complete said process, and it is the main mechanism of production of anemia of this type.

The second mechanism is due to a peripheral hemolysis, of lesser amount, compared to the previous one, and is the early destruction of elements that gain the general circulation, but that due to their morphological and metabolic alterations, are destroyed prematurely.

The fundamental characteristic of megalobastic anemia is macrocytosis, that is, an MCV greater than 95 fl in the vast majority of peripheral elements of the red series.

There are other causes of macrocytosis that are not due to a B12 or folic deficiency and are therefore “non-megalobastic” macrocytosis.

Some of these causes are:

• Increase in normal erythropoiesis (hemolysis, post-bleeding states).
• Increased red cell surface (chronic liver disease, obstructive jaundice, post-splenectomy states).
• Myelodysplasias (dyserytopoietic anemia I, 5q- syndrome, sideroblastic anemia).
• Toxic habits (alcoholism, smoking).
• Hypothyroidism
• Medullary metastasis.
• EPOC
• Electronic artifacts (Cryoagglutinins, conserved blood, hyponatremia).

Vitamin B12 metabolism

Structurally, vitamin B12 is characteristically similar to porphyrin, consisting of a corrinic nucleus containing a cobalt atom and a benzimidazole nucleus.

Chemically, the name of Vitamin B12 corresponds to cyanocobalamin, which is the most stable of all the forms of this compound.

The daily needs of B12 are approximately 2 to 5 ug, and these levels do not vary much with age and gender. The total content of cyanocobalamin in the body is between 2 and 5 mg. Of these, 1 mg. it is found in the liver as a reserve.

Although there is production of B12 by intestinal saprophytic bacteria, it is not very useful and for this reason the diet must contain adequate amounts so as not to fall into a deficit, which occurs after a long time thanks to the enormous liver deposits.

The dietary source of B12 are meat products and their derivatives and to a lesser extent, milk, eggs, and fish. It is almost absent in vegetables.

A normal diet provides between 3 and 30 ug. of B12 per day, of which between 1 and 5 ug are absorbed.

The B12 absorption mechanism is a complex and regulated process that requires several steps:

The first stage consists of the release of cyanocobalamin present in food, facilitated by the acidity of the environment and gastric enzymes.

The second stage consists of the binding of most of the released B12 to proteins, known as fast proteins, which have a much higher affinity for the vitamin than Intrinsic Factor. B12-fast protein complexes pass into the duodenum.

In the third step, which takes place in the duodenum, pancreatic proteases release B12 from fast proteins, and Castle Intrinsic Factor is bound to B12 but not to its analogues. Intrinsic factor is a glycoprotein synthesized by gastric parietal cells of the fundus and cardia. The secretion of intrinsic factor is stimulated by the same effectors that stimulate the secretion of hydrochloric acid (action of vagus, histamine, gastrin, etc); and inhibited by somatostatin and H2 blockers (ranitidine, cimetidine).

Next, the B12-FI complex reaches the distal ileum, where it binds to a membrane receptor, and is internalized in the intestinal cell through an endocytosis process.

Once in the enterocyte, B12 is released from the receptor, binds to its transport protein, Transcobalamin II, and diffuses into plasma.

Transcobalamin II is the only protein that effectively transports B12.

There are 2 more Transcobalamins (I and III) that, although they fix B12, are not capable of delivering it to cells, so they are not considered true transporters.

In humans, there are 2 types of metabolic processes that require B12

- those using methyl cobalamin (conversion of homocysteine ​​to methionine)

- Those using 5-deoxyadenosyl B12 (conversion of methylmalonyl-CoA to succinyl-CoA).

The synthesis of methionine from homocysteine ​​is closely related to the metabolism of folic acid, since it is coupled with the transformation of methyl-tetrahydrofolate (methyl-THF) to methylene-THF.

This metabolite is an essential cofactor for DNA synthesis, so its deficit produces an alteration in DNA synthesis and, at the same time, an accumulation of homocysteine ​​and methyl-THF in plasma, known as the “methyl trap”.

The accumulation of homocysteine ​​is related to vascular pathology and increased predisposition to develop thrombosis.

The methyl trap lowers the levels of S-adenosylmethionine, which contributes to neurological disorders, since this metabolite is necessary for the preservation of myelin.

Folate metabolism

Folic or pteroylglutamic acid is involved in numerous intracellular monocarbon group transfer reactions. Its active form is tetrahydrofolate (THF). In the body and in general in nature, the forms that have more than one molecule of glutamic acid (polyglutamates) are more abundant.

Like B12, intestinal bacteria produce folate, but it is absorbed very little, so the natural source of folic in humans is food.

Although it can be found in any type of food, including meat derivatives, it is widely prevalent in vegetables, especially vegetables (spinach, chard, lettuce, cabbages) and fruits (bananas, melon, citrus), where they are always in the form of polyglutamates .

The daily requirements of a normal adult vary between 50 and 100 ug., And may increase in certain physiological conditions, such as pregnancy or lactation, up to 300 to 500 ug / day. A normal diet contains between 2000 and 4000 ug / day of folate, but unlike what happens with cyanocobalamin, it is a very labile compound and easily destroyed by heat. For this reason, the maximum use of folate occurs when vegetables and fruits are eaten fresh, without cooking.

The main folate reserve organ is the liver, but due to its higher consumption, the reserve capacity is limited and in general, a deficit appears after 3 months of absence of consumption.

In part, folate also accumulates in the cytoplasm of erythroblasts, which remains until more mature stages (erythrocyte) and its measurement is a variable to quantify organic folate stores.

Folic acid circulates in plasma in the form of methyl-THF as monoglutamate.

In the absorption process, polyglutamates are hydrolyzed to monoglutamates, by the action of a hydrolase present in intestinal epithelial cells. Most of the folate is absorbed by passive diffusion and its absorption can be modified by various substances in the diet that can increase or decrease it.

Human milk contains a folate-binding protein that aids in its absorption during lactation. The same effect has vitamin C (ascorbic acid).

In contrast, compounds such as ethanol, barbiturates, phenytoin, and oral contraceptives can decrease it.

In plasma, most of the folic acid is transported nonspecifically bound to albumin. Entry into cells is through the binding of methyl-THF to a specific membrane receptor or through a facilitated diffusion mechanism.

Once inside the cell, it is reconverted to polyglutamate, which is impermeable to the cell membrane and is therefore retained in this form within the cell.

As mentioned above, methylene-THF is involved in an essential step in DNA synthesis, a process that requires B12, as also mentioned above, and a situation that juxtaposes the two nutrients in a common metabolic step.

MEGALOBASTIC ANEMIA

PATHOPHYSIOLOGY

Megalobastic anemia is the result of a maturational disorder of the erythroblasts as a consequence of the deficiency of 2 essential vitamin factors: B12 and folic acid.

In normal hematopoiesis, cells with the capacity to multiply themselves are, most of the time, in the resting phase (phase 0). When mitosis (S) starts, they double their DNA content, divide, and each generated cell reverts to phase 0.

When there is a deficiency of B12 and folic, there is a lengthening of the S phase, which means that, at any moment, there are more cells in S phase trying to duplicate, than in phase 0.

This makes large cells with fine chromatin, typical of nuclear maturation delay, to be seen under the microscope. For its part, hemoglobin synthesis continues its normal rhythm. This makes the distinctive feature of "megaloblasts" (nucleus-cytoplasm dissociation).

Although this phenomenon is mainly expressed in the red series, to a lesser degree it also affects the other series (granulocytes and megakaryocytes), which also show distinctive features of megaloblastic changes.

Although the affectation occurs at all levels, it is more intense at the level of basophilic and polychromatic erythroblasts over orthochromatic ones. In the latter it is easier to observe where a well hemoglobinized cytoplasm (acidophilus) with a still immature nucleus (lax chromatin) can be appreciated.

At the peripheral level, the characteristic macrocytosis (MCV> 95) stands out.

Ovalocytes, presence of cytoplasmic inclusions (basophilic stippling, Howell-Jolly bodies or Cabot's rings), and hypersegmented neutrophils (pleokaryocytes) are also usually observed.

B12 and folic deficiency can also cause cellular alterations in other tissues with high cell turnover such as skin, mucosa, and gastrointestinal epithelium.

ETIOPATHOGENIA

The main causes of B12 deficiency are due to vitamin malabsorption due to gastrointestinal disorders; while the folate deficiency is frequently a deficiency secondary to an inadequate diet.

It is important to remember that in the event of a deficit, the B12 reserves are sufficient to maintain adequate levels for 3 to 4 years, while the folic acid reserves are depleted in 3 to 4 months.

Among all the causes of B12 deficiency, the most important and frequent is the absence of Castle's Intrinsic Factor (FI), due to an alteration of the gastric mucosa, atrophic gastritis. It is characterized by inflammation and subsequent atrophy of the gastric mucosa, which, although it may be multifactorial, has a clear autoimmune basis. There is a progressive decrease and loss of gastric parietal cells, which produce FI, with a marked decrease in the production of hydrochloric acid, pepsin and FI. Atrophic gastritis is characterized by its slow evolution and the almost absence of symptoms, due to which megaloblastic anemia is usually its form of presentation.

Atrophic gastritis is more common in Caucasians, and in Nordic countries and North America. It is also rare in those under 60 years of age.

The autoimmune basis is given by the presence of alterations in humoral immunity (anti-parietal cells or anti-FI), alterations in cellular immunity (deficiency of suppressor T lymphocytes); and due to the fact that it is frequently associated with other autoimmune diseases.

Folate deficiencies, unlike B12, correspond to different causes.

Folate deficiency is mainly due to deficiency causes, which is why it is typical of elderly, malnourished subjects; young people subjected to very extreme and poorly controlled weight loss regimes; indigent subjects with chronic malnutrition and chronic alcoholics. In infants, folic deficiency is seen in those with replacement of milk to goat milk without adequate replacement of the vitamin.

As a second cause, hyperconsumption states such as hyperthyroidism, hemolytic anemias and chronic myeloproliferative syndromes are mentioned.

Folate malabsorption, unlike B12, is more rare and is due to causes that damage the integrity of the intestinal wall or interfere with it (drug).

Symptoms and signs

The clinical picture of megaloblastic anemia is totally similar to that of any other cause of anemia.

Common symptoms such as paleness, fatigue, asthenia, palpitations, dyspnea, are frequent.

The patient's age and knowledge of eating habits are of vital importance here.

Thus, in children and pregnant women, it is most likely that it is a folate deficiency. The presence of alcoholism and / or malnutrition, neoplasms or the intake of certain medications, can alert about the possible folate deficiency.

The appearance of marked megalobastic anemia in the neonatal period should suggest a congenital deficiency of Transcobalamin II.

When the clinical history does not detect these alterations, a B12 deficiency should be considered.

Here, age, the presence of coexisting autoimmune diseases, a family history of anemia and the presence of digestive symptoms (dyspepsia, flatulence, constipation and / or diarrhea) plus neurological symptoms (paresthesia and loss of strength); they are very important to consider.

Neurological manifestations are characteristic of B12 deficiency and never appear in folate deficiency, which is a highly valuable clinical sign.

They consist of a peripheral neuropathy with variable alteration of superficial and deep sensation and are due, as mentioned before, to a demyelination of the lateral and posterior cords of the spinal cord with progressive degeneration of the axons (Subacute Combined Degeneration).

Isolated paresthesias appear progressively in the hands and feet, with slight loss of superficial sensitivity. In more advanced cases, deep sensitivity is altered with loss of muscle strength that, added together, can cause gait disturbances with instability. A positive Romberg sign appears in this state.

Finally, marked muscle weakness, hyperreflexia, spasticity and corresponding disorders of the brain such as behavior and memory changes develop.

It is important to note that up to a third of patients who develop neurological symptoms do so with almost normal hemoglobin values, and it is even more noticeable that neurological symptoms are all the more serious the more normal the hematocrit, hemoglobin and corpuscular volume values ​​are.

In some patients with B12 deficiency it is possible to observe signs such as vitiligo on the skin, corresponding to a broader immune phenomenon associated with atrophic gastritis. It is common to see alterations of the mucosa such as glossitis, inflammation of the lingual mucosa with a papillate and smooth appearance (Hunter atrophic glosses).

Splenomegaly and / or mild hepatomegaly may be found in 10–15% of patients. A characteristic phenotypic trait of patients with atrophic gastritis and secondary megalobastic anemia has been described: white complexion, light eyes, light and fine hair and a tendency to premature baldness.

It is also possible to find clinical signs of underlying autoimmune etiology in these patients: hypo or hyperthyroidism, adrenal insufficiency, diabetes mellitus, or hypoparathyroidism.

Study methodology

The diagnosis of megaloblastic anemia requires first confirming the presence of anemia, and the corresponding deficits and second looking for the underlying cause.

After completing a complete clinical history, we proceed to perform peripheral blood tests.

A complete blood count will confirm the presence of anemia and its macrocytic character (MCV> 95). The presence of macrocytosis can precede the appearance of anemia itself by months to years. In general, the white series and platelets are usually normal, but in some advanced cases, there may be significant leukopenia and platelets, which can confuse the diagnosis with Medullary Aplasia. In these cases, the reticulocyte count can help, as it is very low in Aplasia, normal or only slightly decreased in megaloblastosis.

The examination of the peripheral smear is very important here. With this easy and cheap test, not only the macrocytic character of the anemia is determined, but the characteristic alterations of the red series (ovalocytes, Howell-Jolly bodies, etc.) and the presence of large and hypersegmented neutrophils can be seen.

Histological confirmation of the megaloblastic character of an anemia is only carried out with the bone marrow examination, where an increase in erythropoiesis, megaloblasts in different stages of maturation, myelocytes and giant metamyelocytes and, occasionally, hyperpolypoid giant megacarocytes can be verified.

The ineffective nature of erythropoiesis is responsible for the increase in this series in bone marrow and also responds to an increase in LDH and indirect bilirubin in the blood.

The determination of serum B12 and folate is the next step to confirm this picture.

When the B12 level is less than 100 pg / ml and the serum folate level is less than 3 ng / ml, these deficits can be confirmed. The measurement of intraerythrocyte folate is an increasingly used measure, since it is more stable than serum folate (which can be strongly influenced by the diet), thus more accurately reflects the deposits of folic acid.

The presence of a high number of reticulocytes, as occurs in acute hemolysis, can confuse the condition since it generates macrocytosis and the levels of erythrocyte folic are higher in reticulocytes than in mature red blood cells. Of course, this situation can be easily resolved with a simple observation of the peripheral smear.

It is interesting to note that a not inconsiderable percentage of these patients present iron deficiency together with a B12 or folic deficiency, which can also mask the condition.

Once anemia and B12 and / or folate deficiencies have been confirmed, subsequent examinations should be aimed at finding the underlying cause and thus applying the appropriate therapeutic measures.

In the case of B12 deficiency, it is essential to determine if the cause is an IF deficiency secondary to atrophic gastritis or another cause.

The B12 absorption test in the presence or absence of FI (Schilling's Test) is a relatively simple test that can be used safely, even in patients who have received compound with B12, in whom the hematological picture may be masked.

The determination of antibodies against FI is another test of great value. Unfortunately, only slightly more than half of patients with atrophic gastritis are positive for this test. There are 2 types of antibodies, blockers or type I (they prevent FI-B12 binding) and precipitants or type II, (they inactivate FI or FI-B12 complex, preventing its binding to receptors in the ileum). The most important from the pathological and diagnostic point of view are those of type I, since their presence is exceptional in another autoimmune disease that is not atrophic gastritis. The presence of these antibodies can precede the appearance of a B12 deficiency by months and their positivity confirms the presence of the pathology and makes the performance of the Schilling test unnecessary.

The specificity of the anti-parietal cell antibodies is detected more frequently than the previous ones (> 80%), but their specificity is lower and they can also be observed in the presence of other autoimmune diseases.

Finally, the histological examination of the gastric mucosa, through a biopsy obtained by gastrofibroscopy. This examination is very important, since the observation of parietal and main cells excludes the diagnosis.

Other tests may also be performed to confirm malabsorption, such as a gastrointestinal X-ray examination, stool fat analysis, jejunal biopsy, and stool screening for serial parasites.

Treatment consists of supplementation of deficient nutrients and treatment of the underlying disorder.

The supplementation will seek to correct anemia and epithelial disorders, reduce neurological disorders or prevent their appearance and normalize B12 and folic deposits.

There are commercially B12 supplements in the form of cyanocobalamin or hydroxycobalamin, which, once in the body, are transformed into its active form.

In general, administration is done parenterally, in different regimens, but starting with a large dose (eg, 1000ug / day for one to two weeks), followed by a long-term maintenance dose, depending on the underlying cause.

The effectiveness of the treatment is controlled by the response in red blood cells and the reticulocyte peak that is observed at approximately 7-10 days.

In pernicious anemia, the administration of B12 corrects the hematological disorders, but has no action on atrophic gastritis or on neurological lesions already established.

Atrophic gastritis lacks effective treatment, so the administration of B12 should be indefinite (maintenance) and periodic surveillance is imperative given the possibility of malignant transformation of this type of lesion into gastric carcinoma. 5 to 10% of patients diagnosed with gastric atrophy develop gastric carcinoma and the possibility of developing it is three times higher than the group of the same age and sex without atrophic gastritis.

With regard to folic acid supplementation, it should be noted that a megalobastic anemia due to cyanocobalamin deficiency can respond to high doses of folic acid, but not vice versa. For this reason, the administration of folates alone in a combined deficit can correct hemopoietic disorders, but not neurological ones, which may aggravate a subacute combined myelitis picture or facilitate its appearance.

The therapeutic administration of folic is generally done orally.

In patients undergoing cytostatic treatments with folate antagonists (methotrexate), parenteral administration of pollen is preferred.

Finally, prophylactic treatment with oral folate is recommended in states of hyperconsumption (pregnant women, chronic hemolysis, malnourished elderly, hemodialysis patients, etc.).