Danielle M. Townsley and Griffin P. Rodgers
Besides iron deficiency, shortages of vitamin B12 (cobalamin) and folate are the most common nutritional causes of anemia. The frequencies of these deficiencies are highly dependent upon the population under study. Because vitamin B12 deficiency usually develops as a consequence of insidious malabsorption that occurs over many years, it becomes more prevalent with advancing age. Because folate deficiency is largely a consequence of inadequate dietary folate, it is most prevalent in populations at risk for malnutrition or areas where the food is unfortified.
VITAMIN REQUIREMENTS, SOURCES, AND STORES
To avoid clinically apparent ill effects, the daily adult requirement for vitamin B12 is 1 to 3 µg and for folic acid ~ 200 µg (Table 2.1).1,2 However, recent information indicates that between 4 and 7 µg/day of B12 is necessary to prevent biochemical changes secondary to a limiting supply of the vitamin.3 This suggests that the current recommended dietary allowance (RDA) of 2.4 µg/day may be insufficient. The daily requirement for folate has been more easily met since 1996, when the U.S. Food and Drug Administration mandated that all grains be fortified with the vitamin in order to reduce the risk of neural tube defects in developing fetuses.4
Bacteria in the gut of herbivorous animals synthesize vitamin B12 and supply it to their hosts, who in turn supply it to humans in the form of meat. There is no vitamin B12 in plant products other than that attributable to bacterial contamination. Plants synthesize folic acid and provide it to man directly in fruits and vegetables and indirectly in meat from herbivores.
The human body normally stores 2 to 3 months’ supply of folic acid, although marginally nourished patients, such as chronic alcoholics, may have stores that can be depleted much sooner.5 In contrast, body stores of vitamin B12 are normally sufficient for 5 to 10 years (Table 2.1).
METABOLIC ROLES OF FOLATE AND VITAMIN B12
The metabolic roles of folate and B12 are closely interrelated (Fig. 2.1). Folate derivatives are essential cofactors in thymidylate synthesis, which is a rate-limiting step in the synthesis of DNA. RNA synthesis, however, is not dependent upon folate. Therefore, deficiency of folate limits gene transcription but not RNA translation, retarding cell division but not cytoplasmic protein synthesis. This leads to the typical cytonuclear dissociation of maturation characteristic of megaloblastic hematopoiesis. Because cobalamin supports the recycling of folate, vitamin B12 deficiency causes megaloblastic changes by restricting the folate supply. This restriction can be at least partially overcome by increasing dietary folate, allowing the hematopoietic effects of cobalamin deficiency to be ameliorated by high doses of folic acid. In contrast, the hematopoietic effects of folate deficiency cannot be overcome by treatment with vitamin B12.
FIGURE 2.1 Metabolic pathways involving folic acid and vitamin B12 (cobalamin).
Cobalamin is also necessary in the pathway leading to the synthesis of S-adenosyl-methionine, which is the only donor of methyl groups for numerous reactions in the brain involving proteins, membrane phospholipids, and neurotransmitters.6 Presumably this explains the frequent neuropsychiatric signs and symptoms associated with vitamin B12 deficiency.7 Folate is not involved in these reactions and cannot reverse the neuropsychiatric deficits caused by vitamin B12 deficiency. However, methyltetrahydrofolic acid is the methyl donor for the synthesis of methionine, the precursor of S-adenosyl-methionine. Therefore, folate deficiency may restrict the synthesis of S-adenosyl-methionine and produce some neuropsychiatric effects as well, although this is rare.8
DEVELOPMENT OF VITAMIN B12 DEFICIENCY
Cobalamin deficiency is rarely caused by inadequate intake or increased utilization of the vitamin (Table 2.2). This is in part due to the pronounced enterohepatic circulation of cobalamin. Although strict vegetarians become depleted of vitamin B12, vegetables often contain sufficient bacteria to provide a marginally adequate supply. A developing fetus shunts cobalamin from its mother, placing her at risk of deficiency, particularly if her baseline stores are low. Rarely, intestinal parasites can induce deficiency; for example the fish tapeworm, Diphyllobothrium latum, competes with the host for cobalamin.9Acutely, cobalamin metabolism can be disrupted by nitrous oxide anesthesia and can induce a rapid, usually transient, megaloblastic anemia.10 However, fatalities and severe neuropsychiatric damage have been associated with chronic administration in patients and recreational use of nitrous oxide.11 There is recent evidence that prolonged use of metformin in diabetic patients is associated with reduced serum B12 levels, although the mechanism is unknown.12
Far more commonly, defects in any of the three levels of the gastrointestinal tract can lead to vitamin B12 malabsorption: the fundus of the stomach, the pancreas, or the small bowel. Obviously surgical removal or bypass of any of these regions leads to B12 malabsorption.13 Bariatric surgery is becoming more prevalent and subsequently an important risk factor for developing B12 deficiency. Otherwise the etiology is inflammatory.
Stomach: In the stomach, food (protein)-bound vitamin B12 must be freed by digestion with pepsin and bound to “R-proteins,” which is a generic term for proteins that bind B12.1 The parietal cells in the fundus secrete both the acid necessary for this digestion and intrinsic factor, the protein to which cobalamin is later transferred in the alkaline duodenum. Therefore, any process that damages the parietal cells can lead to vitamin B12 malabsorption and eventually deficiency. The most common cause is autoimmune atrophic gastritis, which increases in prevalence with age and is sometimes associated with other autoimmune diseases, such as thyroiditis. Helicobacter pylori, however, which typically causes antral gastritis, can occasionally also infect the fundus.14,15 Proton pump inhibitors induce chronic hypochlorhydria but rarely cause clinically significant B12 malabsorption. Parodoxically, the hypersecretion of acid in the Zollinger-Ellison syndrome leads to B12 malabsorption by acidifying the small bowel, which must remain alkaline for the transfer of B12 from the R-binders to intrinsic factor. Antibodies to intrinsic factor, as in the case of pernicious anemia, will also lead to a reduction of vitamin B12-intrinsic factor complexes necessary for absorption in the small bowel.
Pancreas: Deficiency of pancreatic enzymes impairs the digestion of R-binders in the small bowel and therefore the release of B12 to intrinsic factor. Although pancreatic insufficiency causes cobalamin malabsorption, it rarely is significant enough to become clinically apparent.
Small bowel: Vitamin B12–intrinsic factor complexes are endocytosed by the mucosa of the terminal ileum. Inflammatory bowel disease or particularly extensive celiac or tropical sprue will interfere with this process.16 Bacterial overgrowth in the small bowel, especially common in the elderly, competes for B12 and makes it less available for absorption.17 HIV infection is sometimes also associated with B12 malabsorption, especially in the presence of chronic diarrhea.
DEVELOPMENT OF FOLIC ACID DEFICIENCY
A diet poor in fresh vegetables is a major cause of folic acid deficiency (Table 2.3). Cooked vegetables and meat are less satisfactory sources because cooking destroys much of the folate. (This is less of a problem for vitamin B12.) The other major causes are gastrointestinal diseases that affect the jejunum, where folic acid is absorbed, and conditions such as pregnancy that increase folate requirements. Ethanol abuse and several chronic medications (Table 2.3) lead to folate deficiency by interrupting folate metabolism or inhibiting its absorption
PATIENT POPULATIONS AT RISK
Vitamin B12 deficiency due to lack of intrinsic factor (“pernicious anemia”) is sometimes believed to be limited to elderly patients of European descent. In this population the median age at presentation is almost 70 years.
Nevertheless, intrinsic factor deficiency may be almost as prevalent in African Americans and Latinos, who tend to present with vitamin B12 deficiency a decade earlier. 18,19
Older patients are actually more likely to become B12 deficient from achlorhydria or small bowel bacterial overgrowth than from lack of intrinsic factor.15,20
Whereas cobalamin deficiency is generally found in older age groups,21 folic acid deficiency is likely to occur in any patient who has an inadequate diet or who has an increased need for the vitamin, e.g., during pregnancy or hemolytic anemia (Table 2.3). Folate deficiency secondary to poor nutrition is generally rare in the United States since the introduction of mandatory folate fortification of cereal grains. A recent population study among centenarians indicates that less than 6% of the very old have low RBC folate levels.22
CLINICAL PRESENTATION
The clinical presentation of vitamin B12 and folic acid deficiency covers a wide range (Table 2.4). The investigation of any new neuropsychiatric changes should include an evaluation of folate and evaluation of vitamin B12 and folate status even in the absence of hematologic signs of a deficiency.7 Changes in the tongue mucosa and mouth angle stomatitis may be the earliest sign of folate deficiency on physical exam.23
LABORATORY EVALUATION
Hematologic Abnormalities
Macrocytosis develops before anemia when either folic acid or vitamin B12 is limiting (Table 2.5).5,24 With the advent of automated blood cell analyzers, isolated macrocytosis has become a typical presentation of deficiencies of either vitamin, although other causes of macrocytosis are more common (Table 2.6).25 An unexplained rise in mean cell volume (MCV) of 5 fL or more even within the normal range should also attract suspicion. However, the macrocytosis may be masked if the patient is also iron deficient or has a thalassemic trait. At hemoglobin concentrations below ~10 g/dL, B12 or folate deficiency leads to elevations of serum lactate dehydrogenase, which can become quite high.5,24 This results from marked intramedullary death of developing red cells and a shortening of the circulating red cell life span but can mislead the clinician to suspect metastatic disease or a primary hemolytic anemia.
The earliest change in the peripheral blood caused by folate or vitamin B12 deficiency is hypersegmentation of the neutrophils, which can be easily overlooked unless a blood smear is carefully examined. Finding even 5% of neutrophils with five lobes or just 1% with six lobes is highly suggestive of a deficiency, although this could also be seen with myelodysplasia. In advanced deficiencies characterized by severe anemia, pancytopenia can develop.
There is rarely if ever a need to perform a bone marrow examination in the evaluation of vitamin B12 and folic acid status. The megaloblastic changes in the marrow are identical in both deficiencies and are variable in intensity. A marrow examination cannot rule out myelodysplasia or even a smoldering leukemic process until cobalamin and folate deficiencies have been excluded first.
Serum Vitamin Concentrations
When vitamin B12 and folate deficiency is suspected, a common diagnostic starting point is measurement of the serum concentrations of the vitamins, which should be done after the patient has been fasting. The results, however, can be difficult to interpret (Table 2.7).
Serum folic acid does not reliably reflect the body’s supply of the vitamin, unless it is consistently below ~3 ng/mL, and even then it does not distinguish between negative balance and actual tissue deficiency.26 In general, the serum folate level reflects recent folate intake, and red cell folate is a better measurement of tissue folate stores. Because red cell folate is packaged at the time the cell is made and remains in the cell throughout its 3 to 4 month lifespan, the measured mean value may fail to reflect relatively recent reductions in dietary folate. Furthermore, the reproducibility of assays for red cell folate is relatively poor so that borderline values can be misleading.26,27 To complicate matters further, red cell folate can be reduced by vitamin B12 deficiency and lead to an erroneous diagnosis (Table 2.7).
Interpreting serum vitamin B12 levels can also be problematic because occasionally serum B12 can be decreased in rare cases of folate deficiency.1 A more frequent problem, similar to that with folic acid, relates to uncertainties in the physiologic levels of the vitamin. The “normal range” of serum cobalamin typically extends down to 200 pg/mL, because healthy, non-anemic donors occasionally have B12 concentrations that are this low. However, patients who are truly deficient in vitamin B12 can have serum cobalamin levels as high as 300 pg/mL and maybe higher.26,28 The reason for this discrepancy is that the total cobalamin is measured, rather than just the vitamin B12 bound to transcobalamin, which is the metabolically available B12 but represents only ~20% of the total serum vitamin. Therefore, individuals who have relatively low concentrations of transcobalamin 1, which binds the other ~80% of the serum vitamin B12, can have alarmingly low B12 levels (<100 pg/mL) without any harmful effect because they have adequate amounts of B12 bound to transcobalamin 2, which is necessary for transfer of B12 to hematopoietic precursors. Occasionally, healthy patients have been described with low transcobalamin 1 levels.29 Transcobalamin 1 can also be low in patients with multiple myeloma.30 Such patients will be easier to identify when tests become available to measure B12 specifically bound to transcobalamin rather than total serum B12.31
Serum Methylmalonic Acid and Homocysteine
Measurements of methylmalonic acid (MMA) and homocysteine (Hcy), though they are more expensive than the vitamin assays, answer the question of vitamin deficiency more reliably.32 Although these metabolites can be elevated for other reasons (Table 2.8), if these causes are excluded, these metabolites become specific reflections of vitamin B12 and folate depletion at the tissue level.19,32 Usually MMA and Hcy both become elevated when B12 is the limiting factor, whereas only Hcy becomes elevated when folate is limiting. However, in 1% to 2% of cases of vitamin B12 deficiency, only Hcy will be elevated, whereas in ~10% of cases of folate deficiency MMA will be high. Therefore, elevated Hcy alone does not always differentiate B12 and folate deficiency but makes folate deficiency more likely.
Although normal levels of MMA and Hcy were reported to exclude a metabolic effect from B12 deficiency, the negative predictive value of these assays has been questioned because of reports of patients with normal metabolites who clearly respond to vitamin B12.19,33
Therapeutic Trial
If the laboratory evaluation is impossible because resources are lacking or evaluation is inconclusive, a therapeutic trial can be diagnostic if a single vitamin is given at a time. Vitamin B12 should be given first because it will do nothing for folic acid deficiency, whereas replacement with folic acid will improve the anemia secondary to B12 deficiency but not the neuropathic changes. The response to treatment can be judged by following the reticulocyte count and hemoglobin (see below). A more expensive way is to remeasure Hcy or MMA levels 2 to 5 days after one or the other of the vitamins has been administered. The metabolites will fall only in response to replacement of the deficient vitamin.32
DETERMINING THE CAUSE OF B12 OR FOLATE DEFICIENCY
The etiology of folate deficiency must always be determined because virtually all causes are either preventable or treatable. If the patient appears to have an adequate diet, a gastrointestinal evaluation is indicated to search for the underlying causes shown in Table 2.3.
In contrast, if vitamin B12 is deficient and the patient is not a vegan and there are no symptoms of gastrointestinal disease, an argument can be made to take the evaluation no further and simply to treat the individual with the vitamin.
If the patient and/or his physician feels compelled to confirm that the pathology lies in the stomach, however, a test for anti-intrinsic factor antibodies should be done.34 A positive test is diagnostic of pernicious anemia and is found in half the cases. Anti-parietal cell antibodies are more common but are also found in a small percentage of normal individuals. Demonstrating an elevation in serum gastrin also strongly supports the diagnosis of gastric atrophy.35
If the patient is unusually young to have achlorhydria or pernicious anemia (e.g., <50 years old or younger if African American), a gastrointestinal evaluation is indicated (Table 2.2).18 In the past, the Schilling test was used in an attempt to detect vitamin B12 malabsorption, but Schilling tests are no longer performed because of lack of a commercial source for radiolabeled cobalamin and the realization that this test also often gives misleading results. Because the incidence of many malignancies, especially gastric cancer, is slightly higher in patients with pernicious anemia than in age- and sex-matched controls, following the patient for any signs of gastrointestinal blood loss is wise, although more aggressive surveillance is not indicated.36
TREATMENT/RESPONSE
There are two treatment goals: replacement of the deficient vitamin and correction of the cause of the deficiency. The first goal is always achievable; the second may not be. Treatment should always be given if the clinical presentation is suspicious, even if the laboratory data are confusing, because the laboratory data are not totally sensitive and the consequences of undertreating can be devastating.32,33 The current evidence indicates that there is no benefit to further supplementation with vitamin B12 or folate if a deficiency is nonexistent. For example, elevated levels of Hcy have been associated with an increased risk of vascular thrombosis, however, there is no role for supplementing vitamin B12 or folate in a sole attempt to decrease Hcy levels and subsequent vascular events.37,38
In the past, vitamin B12 was always given intramuscularly in North America, although the oral route was used in Sweden.39 Recently, however, the effectiveness of oral B12 was demonstrated in two randomized, controlled clinical trials.40,41 This works even in patients with pernicious anemia because approximately 1% of any oral dose of vitamin B12 is absorbed by simple diffusion across the mucosa. So the recommended daily dose of 1 to 2 mg vitamin B12 results in the absorption of ~ 10 to 20 µg, which is much more than the daily requirement. A caveat, however, is that 1 mg tablets of vitamin B12 are sometimes difficult to find in hospital pharmacies.
Intramuscular B12, of course, is perfectly acceptable if it is more practical for a patient, and this route would still be preferred by most physicians to treat a patient with neurologic symptoms. Although there are a variety of accepted regimens, daily injections of 50 to 100 µg should be given for a week, followed by weekly injections for a month, and then monthly injections of 1 mg. For most patients with vitamin B12 deficiency, lifelong treatment is required because the underlying cause is not reversible.
The usual dose of oral folic acid is 1 mg/day (Table 2.1). This is ample even during pregnancy or chronic hemolysis. If the deficiency is dietary, replacement with folic acid should continue until the diet has become adequate. A month of daily folic acid should be sufficient to replenish body stores. If the etiology of the deficiency is small bowel dysfunction, higher doses for longer periods of time may be necessary. It is important to rule out concomitant vitamin B12 deficiency prior to replenishing folate because anemia may improve but the neurologic symptoms due to vitamin B12 deficiency can progress and the diagnosis missed.
The response to correction of vitamin B12 or folate deficiency is the same:
Mental changes and tongue soreness improve almost immediately after starting to replace the deficient vitamin. 1
After 4 to 5 days, reticulocytosis appears and may elevate the MCV even further.
Soon thereafter, the hemoglobin concentration begins to rise.
Neuropathic abnormalities, such as paresthesias, improve slowly, over several months, but may never disappear entirely if they have been long-standing.
If the hematologic response is blunted, additional etiologies for the anemia should be sought. It is not unusual for iron deficiency to accompany folate or vitamin B12 deficiency. An underlying anemia of chronic disease is always a possibility.
References