Chapter 12 Megaloblastic Anemias

Figure 12-1 COMPONENTS AND MECHANISM OF COBALAMIN ABSORPTION.

Cbl, Cobalamin; D. latum, Diphyllobothrium latum; HCl, hydrochloric acid; IF, intrinsic factor; R-Cbl, R-protein bound cobalamin; TCII, transcobalamin II.

Figure 12-2 CELLULAR UPTAKE AND INTRACELLULAR REACTIONS INVOLVING COBALAMIN.

A large family of natural and synthetic cobalamins can be generated when the cyanide (CN) moiety (upper axial ligand in cyanocobalamin) is replaced. On exposure to light, CN is gradually lost from cyanocobalamin, with the production of hydroxocobalamin. In vivo substitutions include the replacement of hydroxocobalamin or cyanocobalamin by a 5′-deoxyadenosyl group attached by a covalent bond, giving rise to adenosylcobalamin (AdoCbl). Methylcobalamin (MeCbl) is the main form in plasma. In vivo, 5-methyl-tetrahydrofolate readily donates its methyl group to cob(I)alamin in a reaction involving methionine synthase to form methylcobalamin. The approximate loci for defects in cobalamin mutants, cblA to cblG, are shown. MMCoA mutase, Methylmalonyl-CoA mutase; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine.

Figure 12-3 MODEL FOR HOW THE CELL SENSES FOLATE DEFICIENCY AND RESPONDS BY UPREGULATING FOLATE RECEPTORS.

Note how this model links perturbed folate metabolism, ribonucleic acid (RNA)–protein interaction, and coordinated translational regulation of folate receptor to optimize cellular folate uptake and restore folate homeostasis. The prominent red arrow highlights the critical role of heterogeneous nuclear ribonucleoprotein E1 (hnRNP-E1) as a candidate sensor of cellular folate deficiency. A, Reduced folate availability results in inactivation of methionine synthase and intracellular homocysteine buildup, which induces a direct posttranslational homocysteinylation of hnRNP-E1 via targeted homocysteine-S-S-cysteine mixed disulfide bonds; this results in the unmasking of a high-affinity folate receptor messenger RNA (mRNA) cis-element binding site and leads to increased translation of folate receptor-α. The net effect is a homeostatic response that aims to restore intracellular folate concentrations to normal by upregulating cell surface folate receptor. Folate repletion reactivates methionine synthase, which converts homocysteine to methionine. Methionine has no effect on the RNA-protein interaction that leads to reduced folate receptor-α synthesis and its downregulation.³⁷ (Note: Other metabolic pathways involving homocysteine^41,42 are not included.) B, A proposed mechanism for the unmasking of a cryptic mRNA binding site in hnRNP-E1 following the covalent binding of L-homocysteine, through the replacement of one (of many potential) cysteine disulfide bonds by protein-cysteine-S-S-homocysteine mixed disulfide bonds. 5′-UTR, 5′ Untranslated region.

(From Tang YS, Khan RA, Zhang Y, et al: Incrimination of heterogeneous nuclear ribonucleoprotein E1 (hnRNP-E1) as a candidate sensor of physiological folate deficiency. J Biol Chem 286:39100, 2011.)

Figure 12-4 MEGALOBLASTIC ANEMIA.

The peripheral smear (A) exhibits macro-ovalocytosis and hypersegmented polys (inset). The bone marrow aspirate (B) shows megaloblastic changes in both granulopoiesis and erythropoiesis. The biopsy (C) is hypercellular and shows sheets of immature erythroid precursors with the appearance of a high mitotic rate. These can mimic acute erythroleukemia or even metastatic tumor cells. Details from the cells in the aspirate (D) compared with normal hematopoiesis at same magnification (E). Note the giant metamyelocyte and band form. In megaloblastic anemia, megakaryocytes also have nuclear atypica, including abnormal nuclear segmentation (F).

Table 12-1 Stepwise Approach to the Diagnosis of Cobalamin and Folate Deficiency

* Serum cobalamin levels: abnormally low, less than 200 pg/mL; clinically relevant low-normal range, 200 to 300 pg/mL.

† Serum folate levels: abnormally low, less than 2 ng/mL; clinically relevant low-normal range, 2 to 4 ng/mL.

‡ Any frozen-over sample from serum folate/cobalamin determination can be subjected to metabolite tests.

Serum Homocysteine and Methylmalonic Acid Levels in Cobalamin and Folate Deficiencies

The combined use of homocysteine and methylmalonic acid (MMA) levels can differentiate cobalamin from folate deficiency, because most patients with folate deficiency have normal MMA levels, and the remainder have only mild elevations.² These two tests are useful diagnostically. The abnormally high levels of metabolites return to normal only when the patient receives replacement with the appropriate (deficient) vitamin. A positive response to cobalamin, documented by falling levels of homocysteine and MMA, is evidence of cobalamin deficiency. Conversely, therapy with folate results in a decrease in the isolated homocysteine level if folate deficiency is present.² Indeed, because several variables that are not related to vitamin deficiency (such as age, mild renal dysfunction) can falsely elevate serum homocysteine and MMA levels, if there is ambiguity, proof of vitamin deficiency would require clear-cut demonstration of a reduction in metabolite levels after specific vitamin supplementation.^2,³

Modified Therapeutic Trials

The traditional therapeutic trial using physiologic doses of vitamins (100 mcg of folate or 1 mcg of cobalamin given daily while monitoring the reticulocyte response)¹ has given way to a modified therapeutic trial. Rather than making the diagnosis of a deficiency, the intention is often to confirm the clinical suspicion that the patient does not have deficiency. This can be demonstrated by lack of response to full replacement doses of both vitamins (1 mg of folic acid orally for 10 days and 1 mg of cobalamin intramuscularly or subcutaneously daily for 10 days). Clinical scenarios in which such trials may be applicable (after drawing blood for serum cobalamin and folate levels) are as follows:

1. There is a clinical suspicion that the underlying disease is not caused by a vitamin deficiency, but this idea is not supported by results of clinical, morphologic, and biochemical evaluations. Such conditions include anemia with a megaloblastic bone marrow that may be secondary to chemotherapy, myelodysplastic syndromes, or acute leukemia; when time is of the essence in making the diagnosis; when the levels of cobalamin are likely to be falsely abnormal because of these diseases; or when there is underlying dehydration or renal dysfunction that predictably gives falsely high levels of metabolites.

2. In other situations (i.e., pregnancy, acquired immunodeficiency syndrome [AIDS], or alcoholism) with a multifactorial basis for anemia, the response or lack thereof to full replacement doses can eliminate cobalamin or folate deficiency and thereby narrow the (often extensive) differential diagnosis.

3. In instances when severe anemia with megaloblastosis is clinically obvious and so serious that the physician cannot wait for the results of specific tests for deficiency. Full doses of both vitamins are administered, and if there is a response manifested by brisk reticulocytosis by days 5 to 7, retrospective assignment of the deficiency is based on the results of blood samples drawn before beginning the trial.

In all therapeutic trials, if there is no evidence of response within 10 days, bone marrow aspiration is indicated to identify another primary hematologic disease.

Table 12-2 Serum Cobalamin: False-Positive and False-Negative Test Results

FALSELY LOW SERUM COBALAMIN IN THE ABSENCE OF TRUE COBALAMIN DEFICIENCY

Folate deficiency (one-third of patients)

Multiple myeloma

TCI deficiency

Megadose vitamin C therapy

FALSELY RAISED COBALAMIN LEVELS IN THE PRESENCE OF A TRUE DEFICIENCY*

Cobalamin binders (TCI and II) increased (e.g., myeloproliferative states, hepatomas, and fibrolamellar hepatic tumors)