Absorption

Studies to measure the actual absorption of B₁₂ involve whole-body counting of radiolabeled B₁₂, counting of radiolabeled B₁₂ in the stool, or both. No data are available on whether B₁₂ absorption varies with B₁₂ status, but fractional absorption decreases as the oral dose is increased (Chanarin, 1979). Total absorption increases with increasing intake. Adams and colleagues (1971) measured fractional absorption of radiolabeled cyanocobalamin and reported that nearly 50 percent was retained at a 1-μg dose, 20 percent at a 5-μg dose, and just over 5 percent at a 25-μg dose. The second of two doses of B₁₂ given 4 to 6 hours apart is absorbed as well as the first (Heyssel et al., 1966). When large doses of crystalline B₁₂ are ingested, up to approximately 1 percent of the dose may be absorbed by mass action even in the absence of intrinsic factor (Berlin et al., 1968; Doscherholmen and Hagen, 1957).

Absorption from Food. The approximate percentage absorption of B₁₂ from a few foods is presented in Table 9-1. These values apply to normal, healthy adults. No studies were found on the absorption of B₁₂ from dairy foods or from red meat other than mutton and liver. The absorption efficiency of B₁₂ from liver reportedly was low because of its high B₁₂ content. Although evidence indicates that a B₁₂ content of 1.5 to 2.5 μg/meal saturates ileal receptors and thus limits further absorption (Scott, 1997), absorption of as much as 7 μg in one subject (18 percent) was reported from a serving of liver paste that contained 38 μg of B₁₂ (average absorption was 4.1 μg or 11 percent) (Heyssel et al., 1966).

TABLE 9-1

Percentage Absorption of Vitamin B₁₂ from Foods by Healthy Adults.

Assumptions Used in this Report. Because of the lack of data on dairy foods and most forms of red meat and fish, a conservative adjustment for the bioavailability of naturally occurring B₁₂ is used in this report. In particular, it is assumed that 50 percent of dietary B₁₂ is absorbed by healthy adults with normal gastric function. A smaller fractional absorption would apply, however, if a person consumed a large portion of foods rich in B₁₂. Different levels of absorption are assumed under various conditions, as shown in Table 9-2. Crystalline B₁₂ appears in the diet only in foods that have been fortified with B₁₂, such as breakfast cereals and liquid meal replacements.

TABLE 9-2

Assumed Vitamin B₁₂ Absorption under Different Conditions.

Enterohepatic Circulation

B₁₂ is continually secreted in the bile. In healthy individuals most of this B₁₂ is reabsorbed and available for metabolic functions. El Kholty et al. (1991) demonstrated that the secretion of B₁₂ into the bile averaged 1.0 ± 0.44 nmol/day (1.4 μg/day) in eight cholecystectomized patients, and this represented 55 percent of total corrinoids. If approximately 50 percent of this B₁₂ is assumed to be reabsorbed, the average loss of biliary B₁₂ in the stool would be 0.5 nmol/day (0.7 μg/day). Research with baboons (Green et al., 1982) suggests that the form of B₁₂ present in bile may be absorbed more readily than is cyanocobalamin, but the absorption of both forms was enhanced by intrinsic factor. Both Green and colleagues (1982) and Teo and coworkers (1980) reported data suggesting that bile enhances B₁₂ absorption. However, in the absence of intrinsic factor, essentially all the B₁₂ from the bile is excreted in the stool rather than recirculated. Thus, B₁₂ deficiency develops more rapidly in individuals who have no intrinsic factor or who malabsorb B₁₂ for other reasons than it does in those who become complete vegetarians and thus ingest no B₁₂.

Excretion

If the circulating B₁₂ exceeds the B₁₂ binding capacity of the blood, the excess is excreted in the urine. This typically occurs only after injection of B₁₂. The highest losses of B₁₂ ordinarily occur through the feces. Sources of fecal B₁₂ include unabsorbed B₁₂ from food or bile, desquamated cells, gastric and intestinal secretions, and B₁₂ synthesized by bacteria in the colon. Other losses occur through the skin and metabolic reactions. Fecal (Reizenstein, 1959) and urinary losses (Adams, 1970; Heinrich, 1964; Mollin and Ross, 1952) decrease when B₁₂ stores decrease. Various studies have indicated losses of 0.1 to 0.2 percent of the B₁₂ pool per day (Amin et al., 1980; Boddy and Adams, 1972; Bozian et al., 1963; Heinrich, 1964; Heyssel et al., 1966; Reizenstein et al., 1966) regardless of the size of the store, with the 0.2 percent value generally applicable to those with pernicious anemia.

Hematological Effects of Deficiency

The major cause of clinically observable B₁₂ deficiency is pernicious anemia (see “Pernicious Anemia”). The hematological effects of B₁₂ deficiency are indistinguishable from those of folate deficiency (see Chapter 8). These include pallor of the skin associated with a gradual onset of the common symptoms of anemia, such as diminished energy and exercise tolerance, fatigue, shortness of breath, and palpitations. As in folate deficiency, the underlying mechanism of anemia is an interference with normal deoxyribonucleic acid (DNA) synthesis. This results in megaloblastic change, which causes production of larger-than-normal erythrocytes (macrocytosis). This leads first to an increase in the erythrocyte distribution width index and ultimately to an elevated mean cell volume. Oval macrocytes and other abnormally shaped erythrocytes are present in the blood. Typically, as with folate deficiency, the appearance of hypersegmentation of polymorphonuclear leukocytes precedes the development of macrocytosis. However, the sensitivity of this finding has recently been questioned (Carmel et al., 1996). By the time anemia has become established, there is usually also some degree of neutropenia and thrombocytopenia because the megaloblastic process affects all rapidly dividing bone marrow elements. The hematological complications are completely reversed by treatment with B₁₂.

Neurological Effects of Deficiency

Neurological complications are present in 75 to 90 percent of individuals with clinically observable B₁₂ deficiency and may, in about 25 percent of cases, be the only clinical manifestation of B₁₂ deficiency. Evidence is mounting that the occurrence of neurological complications of B₁₂ deficiency is inversely correlated with the degree of anemia; patients who are less anemic show more prominent neurological complications and vice versa (Healton et al., 1991; Savage et al., 1994a). Neurological manifestations include sensory disturbances in the extremities (tingling and numbness), which are worse in the lower limbs. Vibratory and position sense are particularly affected. Motor disturbances, including abnormalities of gait, also occur. Cognitive changes may occur, ranging from loss of concentration to memory loss, disorientation, and frank dementia, with or without mood changes. In addition, visual disturbances, insomnia, impotency, and impaired bowel and bladder control may develop. The progression of neurological manifestations is variable but generally gradual. Whether neurological complications are reversible after treatment depends on their duration. The neurological complications of B₁₂ deficiency occur at a later stage of depletion than do the indicators considered below and were, therefore, not used for estimating the requirement for B₁₂. Moreover, neurological complications are not currently amenable to easy quantitation nor are they specific to B₁₂ deficiency.

Gastrointestinal Effects of Deficiency

B₁₂ deficiency is also frequently associated with various gastrointestinal complaints, including sore tongue, appetite loss, flatulence, and constipation. Some of these complaints may be related to the underlying gastric disorder in pernicious anemia.

Prevalence of Atrophic Gastritis

Large differences in the prevalence of atrophic gastritis in the elderly, ranging from approximately 10 to 30 percent, have been reported in Australia (Andrews et al., 1967), Missouri (Hurwitz et al., 1997), Scandinavia (Johnsen et al., 1991), and Boston (Krasinski et al., 1986). In the general elderly population, many cases of atrophic gastritis may remain undiagnosed.

Food-Bound B₁₂ Malabsorption

Testing of individuals who have low serum B₁₂ values but who do not have pernicious anemia reveals a substantial proportion with malabsorption of protein-bound B₁₂ (Carmel et al., 1987, 1988; Jones et al., 1987). More importantly, Carmel and coworkers (1988) found that 60 percent of those with neurological, cerebral, or psychological abnormalities malabsorbed food-bound B₁₂. Food-bound malabsorption is found in persons with certain gastric dysfunctions (e.g., hypochlorhydria or achlorhydria with an intact stomach, post-gastric surgery such as Billroth I or II, and postvagotomy with pyloroplasty) and in some persons with initially unexplained low serum B₁₂ (Carmel et al., 1988; Doscherholmen et al., 1983). Suter and colleagues (1991) reported that subjects with atrophic gastritis absorb significantly less B₁₂ than do healthy control subjects but that the difference disappears after antibiotic therapy.

Miller and colleagues (1992) studied the absorption of radiolabeled B₁₂ in patients who had not had gastric surgery but who had low B₁₂ values. All patients with elevated serum gastrin levels absorbed food-bound B₁₂ poorly compared with 21 percent of all those with normal serum gastrin values. In this study normal values were specified as greater than 12 percent absorption of food-bound B₁₂ and greater than 33 percent absorption of free B₁₂ as measured by direct body radioactivity measurements. Control subjects with normal serum B₁₂ values (median 173 pmol/L [234 pg/mL], range 125 to 284 pmol/L [170 to 385 pg/mL]) absorbed 12 to 39 percent of food-bound B₁₂ and 54 to 97 percent of free B₁₂ (median 75 percent). The median age of this group was 61 years (range 49 to 69 years). Available evidence does not indicate that aging or atrophic gastritis increases the amount of B₁₂ that must actually be absorbed to meet the body's needs.

Folate with B₁₂

Although adequate or high folate intake may mitigate the effects of a B₁₂ deficiency on normal blood formation, there is no evidence that folate intake or status changes the requirement for B₁₂.

Vitamin C with B₁₂

Low serum B₁₂ values reported in persons receiving megadoses of vitamin C are likely to be artifacts of the effect of ascorbate on the radioisotope assay for B₁₂ (Herbert et al., 1978)—and thus not a true nutrient-nutrient interaction.

Methods Used to Set the Adequate Intake

An Adequate Intake (AI) is set for the recommended intake for infants. The AI reflects the observed average vitamin B₁₂ intake of infants fed principally with human milk.

Reported values for the concentration of the vitamin in human milk vary widely, partly because of differences in methods of analysis and partly because of differences in maternal B₁₂ status and current intake. Despite high intraindividual diurnal variability within a group of lactating women, no consistent effect on B₁₂ concentration of time of day, breast, or time within a feed has been demonstrated. Thus, casual samples of human milk can be used to represent concentrations for the group (Trugo and Sardinha, 1994). However, the wide intraindividual variability may lead to inaccuracies in reported mean values if the number of individuals sampled is small. Median values are substantially lower than average values (Casterline et al., 1997; Donangelo et al., 1989). Acceptable methods of analysis include Euglena gracilis after pretreatment with papain to release the vitamin from the R protein in milk and radioassays in which the vitamin is released by heating (Areekul et al., 1977; Trugo and Sardinha, 1994). Studies used for estimating the concentration of the vitamin in human milk are limited to those that used one of these two methods.

The single longitudinal study of the change in B₁₂ concentration in human milk over time (Trugo and Sardinha, 1994) suggests somewhat higher concentrations in colostrum than in mature milk (≤ 21 days postpartum) but little change after the first month of lactation.

Ages 0 through 6 Months

The AI for infants ages 0 through 6 months is based on the B₁₂ intake of infants fed human milk. B₁₂ deficiency does not occur in infants fed milk from mothers with adequate B₁₂ status. In samples collected from nine well-nourished Brazilian mothers who were not taking supplements and whose infants were receiving human milk exclusively, the average concentration of the vitamin was 0.42 μg/L at 2 months; this decreased to an average of 0.34 μg/L at 3 months (Trugo and Sardinha, 1994). Milk collected at least 2 months postpartum from 13 unsupplemented American mothers who were vegetarians was lower in B₁₂ content, averaging 0.31 μg/L (Specker et al., 1990). The B₁₂ content of milk in a large group of low-income Brazilian mothers (n = 83) who had received prenatal supplements containing B₁₂ was much higher, averaging 0.91 μg/L after 1 month of lactation (Donangelo et al., 1989). Given that the average concentration at 2 months postpartum of well-nourished mothers whose infants received exclusively human milk was higher than those on vegetarian diets, the higher value of 0.42 μg/L is chosen in order to be sure adequate amounts are available. Using the average human milk volume of 0.78 L/day during the first 6 months and the higher average B₁₂ content of 0.42 μg/L, the AI for B₁₂ for the infant 0 through 6 months of age fed human milk would be 0.33 μg/day, rounded up to 0.4 μg.

Maintenance of Normal Methylmalonic Acid Concentrations. Data on methylmalonic acid (MMA) excretion is also available for infants. An infant may be born with low B₁₂ stores and may consume human milk that is low in B₁₂ if its mother is a vegan (a person who avoids all animal foods) or has untreated pernicious anemia. Such infants begin to show clinical signs of B₁₂ deficiency at about 4 months postpartum. In the study of 13 vegan mothers and their infants, Specker and colleagues (1990) found increased urinary MMA in 2-to 14-month old (mean 7.3) infants predominantly fed human milk when the B₁₂ concentration in human milk was below 0.49 μg/L. Assuming an average volume of human milk consumption of 0.78 L/day during the first 6 months, the infant of a vegan mother would be receiving an average of 0.24 μg/day of B₁₂ (0.31 μg/L × 0.78 L/day).

In these infants, urinary MMA concentrations were strongly correlated with those of their mothers and inversely related to maternal plasma B₁₂ concentrations, supporting the assumption that the elevations in infant urinary MMA were caused by poor maternal B₁₂ status (Specker et al., 1988). Although these infants were probably born with depleted stores of the vitamin, the data suggest that a mean intake of 0.24 μg/day is inadequate to maintain B₁₂ balance in infants.

Clinical signs of B₁₂ deficiency are usually seen if the mother has been a strict vegetarian for at least 3 years. The B₁₂ status of the infant is clearly abnormal by about 4 to 6 months of age. In case studies of infants born to strict vegetarians who were identified because of clinical signs of B₁₂ deficiency, human milk concentrations have been reported to be 0.02 (Hoey et al., 1982), 0.037 (Gambon et al., 1986), 0.032 and 0.042 (Jadhav et al., 1962), 0.051 (Johnson and Roloff, 1982), and 0.085 (Kuhne et al., 1991) μg/L. If clinical signs appear within 9 months in infants consuming milk containing 0.085 μg/L of B₁₂, this intake (approximately 0.07 μg/day) cannot support B₁₂ requirements of the infant during the first year. However, from these data it cannot be determined how far this estimate falls below the average requirement for these infants.

Rate of Depletion of Stores. The liver of a well-nourished newborn infant contains 18 to 22 pmol (25 to 30 μg) of B₁₂ (Baker et al., 1962; Loria et al., 1977; Vaz Pinto et al., 1975). There are no data on liver B₁₂ content at birth in full-term infants born to depleted mothers, only data for two infants who died prematurely (Baker et al., 1962); thus, the utilization of B₁₂ by infants remains speculative.

Summary. The AI for infants ages 0 through 6 months is 0.33 μg/day based on the average concentration of B₁₂ in the milk of mothers with adequate B₁₂ status. This value is rounded up to 0.4 μg/day. The adequacy of this intake is supported by evidence that it is above the intake level that has been associated with increased urinary MMA excretion.

Ages 7 through 12 Months

If the reference body weight ratio method described in Chapter 2 to extrapolate from the AI for B₁₂ for infants ages 0 through 6 months is used, the AI for B₁₂ for the older infants would be 0.5 μg/day after rounding up. This is a somewhat lower value than that obtained from the second method (see Chapter 2) by extrapolating down from the Estimated Average Requirement (EAR) for adults and adjusting for the expected variance to estimate a recommended intake, which results in an AI for B₁₂ of 0.6 μg/day.

In one study of three infants exclusively fed human milk who had clinically observable B₁₂ deficiency caused by low maternal consumption of animal products, one infant was treated parenterally with B₁₂ whereas two infants were treated with small oral B₁₂ doses (Jadhav et al., 1962). At 9 months of age, 0.1 μg/day of oral B₁₂ normalized bone marrow within 5 days in one of the two infants given oral doses and produced profound improvements in behavior by 18 days (after a total of 1.8 μg of B₁₂ had been given). The mother's milk contained 0.032 μg/L. In the second infant, who was 7 months old, 0.1 μg/day of B₁₂ caused abnormal pigmentation to disappear and “an adequate hematologic response.” His mother's milk contained 0.042 μg/L. Although evidence of sustained recovery was not provided, it appears from these limited data that 0.1 μg/day may be adequate to improve clinical and hematological signs of deficiency in infants at this age. However, it is not known whether this level of intake is adequate to sustain normal plasma B₁₂ and MMA concentrations or hematological response.

In the study of infants of vegan mothers (Specker et al., 1990) the mean age of infants was 7.3 months (ranged 2 to 14 months). As discussed, a mean intake of 0.23 μg/day was not adequate to maintain B₁₂ balance in this group as determined by urinary MMA excretion.

B₁₂ AI Summary, Ages 0 through 12 Months

Special Considerations

Infants of vegan mothers should be supplemented with B₁₂ at the AI from birth on the basis of evidence that their stores at birth are low and their mother's milk may supply very small amounts of the vitamin.

Method Used to Estimate the Average Requirement

Only one study is available to provide data regarding B₁₂ status and intake in young children. Plasma MMA was elevated in 11- to 22-month-old (mean 16.8 months) infants of Dutch vegan mothers. The sensitivity of plasma MMA to distinguish the group of infants born to macrobiotic mothers from those born to omnivorous mothers was 85 percent (Schneede et al., 1994). The average intake of B₁₂ by these infants, who were exclusively fed human milk for a mean of 4.8 months and then at least partially fed human milk for 13.6 ± 6.6 (standard deviation) months and fed macrobiotic foods, was 0.3 ± 0.2 μg/day for the first 6 to 16 months compared with 2.9 ± 1.2 μg/day for well-nourished control infants (Dagnelie et al., 1991). These data suggest that an intake of 0.3 μg/day of B₁₂ between 6 and 16 months of age was inadequate to prevent elevated plasma MMA concentrations of infants born to vegan mothers.

No other direct data were found on which to base an Estimated Average Requirement (EAR) for B₁₂ for children or adolescents. In the absence of additional information, EARs and RDAs for children and adolescents have been estimated by using the method described in Chapter 2, which extrapolates down from adult values, and rounded up.

B₁₂ EAR and RDA Summary, Ages 1 through 18 Years

The RDA for B₁₂ is set by assuming a coefficient of variation (CV) of 10 percent (see Chapter 1) because information is not available on the standard deviation of the requirement for B₁₂; the RDA is defined as equal to the EAR plus twice the CV to cover the needs of 97 to 98 percent of the individuals in the group (therefore, for B₁₂ the RDA is 120 percent of the EAR).

Method Used to Estimate the Average Requirement

No single indicator was judged to be a sufficient basis for deriving an EAR for adults. It was not deemed appropriate to base the EAR on an examination limited to studies that provided data on mean cell volume (MCV) or serum B₁₂ or any other single laboratory value. Data on men and women were examined together because of small numbers. Three general approaches were considered to derive the EAR for adults: determination of the amount of B₁₂ needed to maintain adequate hematological status (as measured by stable hemoglobin value, normal MCV, and normal reticulocyte response) and serum B₁₂ values in persons with pernicious anemia or with known intakes that were very low in dietary B₁₂; use of daily B₁₂ turnover to estimate the amount of B₁₂ needed to maintain body stores at a specified level; and estimation of the dietary B₁₂ intake by healthy adults that corresponds to adequate serum values of B₁₂ and of MMA.

The first approach was chosen as the primary method for deriving an EAR because it is the only approach for which there are sufficient and reliable data for estimating need. A low serum B₁₂ value in persons with pernicious anemia was assumed to indicate incomplete response to treatment.

Primary Criterion: Maintenance of Hematological Status and Serum B₁₂ Values. The primary method used to derive the EAR for adults estimates the amount of B₁₂ needed for the maintenance of hematological status and serum B₁₂ values, primarily by using data derived from patients with pernicious anemia in remission. Data from studies of vegetarians were also examined to determine whether they provided information on levels of B₁₂ intake needed to maintain hematological status. In some cases, neurological manifestations may be the earliest clinical sign of low B₁₂ values (Beck, 1991; Karnaze and Carmel, 1990; Lindenbaum et al., 1988; Martin et al., 1992). Assumptions that were integral to the application of this method are shown in Box 9-1.

BOX 9-1

Assumptions Made in Estimating the Amount of Vitamin B₁₂ Needed for Maintenance of Hematological Status and Serum Vitamin B₁₂ Values. Maintenance of hematological status requires a relatively stable hemoglobin value upon administration of B₁₂ and a normal (more...)

In brief, this method involves estimating the amount of B₁₂ required daily to maintain hematological and serum B₁₂ status of individuals with pernicious anemia in remission; subtracting the amount of endogenous B₁₂ lost from the bile in excess of that lost by a healthy individual; and, because the value is to be used for individuals with normal ability to absorb B₁₂ from food, correcting for bioavailability. The result is shown in Box 9-2.

BOX 9-2

Steps Used to Estimate the Vitamin B₁₂ Requirement by Using Data Obtained from Subjects with Pernicious Anemia.

The following studies provide the basis for the estimate used in Step 1. These studies do not provide ideal data on which to base an EAR, but they bracket the requirement by providing values that are obviously too low or too high to meet the needs of 50 percent of the individuals in an age group.

Studies of Patients with Pernicious Anemia. Darby and coworkers (1958) studied the effects of various intramuscular (IM) doses of B₁₂ in 20 subjects with pernicious anemia who had not previously been treated or who were in relapse. The diagnosis of pernicious anemia had been based on the clinical history and on the findings of macrocytic anemia, megaloblastic hyperplasia of bone marrow, histamine-fast achlorhydria, and a negative radiological examination of the gastrointestinal tract. These diagnoses were not made based on results of the Schilling test, first published as a method in 1953 (Schilling, 1953). The extent of the disease differed among the subjects; 14 had neurological manifestations. Of the 18 subjects who received doses of 1 μg/day of B₁₂ or less for 2 weeks, 5 or fewer responded satisfactorily according to the standards used for erythrocytes (Isaacs et al., 1938) and reticulocytes (Isaacs and Friedman, 1938). At B₁₂ dosages of less than 0.5 μg/day, no patient met those standards. Dosages used for maintenance were increased to 1 to 4 μg/day for a period of months to years. MCVs greater than 100 were considered macrocytic. No reticulocyte counts or serum B₁₂ values were reported. According to the authors' interpretation, the data indicated that subjects achieved and maintained maximum erythropoiesis as indicated in Table 9-5. Approximately half (4 of 7) did so at a B₁₂ intake of 1.4 μg/day IM.

TABLE 9-5

Effectiveness of Intramuscular Vitamin B₁₂ Doses for Maintenance of Maximum Erythropoiesis.

Results of other studies of patients with pernicious anemia are presented in Table 9-6. The short-term study by Hansen and Weinfeld (1962) used relatively high B₁₂ doses to restore normal status but did not assess maintenance requirement. The long-term studies by Bastrup-Madsen et al. (1983) and Lindenbaum et al. (1990) used different dosages and methods of reporting that make it impossible to draw precise conclusions. Nonetheless, the results indicate that 0.8 to 1.0 μg/day of B₁₂ IM will maintain normal hematological, serum B₁₂, and serum metabolite status in nearly half of the individuals over time and that 1.7 μg will maintain it in all individuals. The study conducted by Best and colleagues (1956) was designed to determine the effective dosage of intrinsic factor concentrates, not to estimate the B₁₂ requirement, but it suggests that 1.4 μg of B₁₂ exceeds the requirement for absorbed B₁₂ in most of the subjects tested. The often-cited study of Sullivan and Herbert (1965) was interpreted as providing evidence that 0.1 μg/day of B₁₂ was not sufficient for treating pernicious anemia and maintaining adequate B₁₂ status. Similarly, the 0.6 to 0.7 μg/day of B₁₂ supplied IM in the study by Will and coworkers (1959) was also judged too low to maintain a normal serum B₁₂ concentration.

TABLE 9-6

Other Studies of Subjects with Pernicious Anemia Considered in Setting the Estimated Average Requirement for Vitamin B₁₂ for Adults.

The study by Darby and colleagues (1958), which indicates an average requirement in such patients of approximately 1.5 μg, is supported by the supplementary data from the other studies described in Table 9-6. These studies provide support for a physiological average requirement of 1.0 μg/day of B₁₂ after adjustment for the extra loss of B₁₂ by subjects with pernicious anemia (0.5 μg/day) (Step 2 in Box 9-2). Adjusting for incomplete absorption of B₁₂ from food of 50 percent (Step 3) converts this value to an EAR for B₁₂ of 2.0 μg/day.

Studies of Individuals with Low B₁₂ Intake. Studies of individuals with low B₁₂ intake were examined to determine whether these reports (Table 9-7) supported the findings for subjects with pernicious anemia. Because B₁₂ is not a component of plant foods, diets containing little or no animal food may lead to B₁₂ deficiency. Deficiency develops slowly because of efficient reabsorption of biliary B₁₂. It is also possible but not certain that vegans consume some B₁₂ from animal products that contaminate plant food or from bacterial action. Studies of vegetarians generally have not analyzed the B₁₂ content of the food, and accurate data are not available for some of the foods (e.g., certain algae) consumed by vegetarians. Without actual analyses it is not clear what B₁₂ content should be assumed for vegans.

TABLE 9-7

Studies of Individuals with Low Vitamin B₁₂ Intake Considered in Setting the Estimated Average Requirement for B₁₂ for Adults.

The studies covered by Table 9-7 suggest that the B₁₂ requirement is higher than the amounts reported to be consumed by the subjects and more than that provided by the treatments that were described. In three studies (Baker and Mathan, 1981; Jathar et al., 1975; Winawer et al., 1967), all adults required more than 1 μg/day of B₁₂ by mouth. Two studies (Narayanan et al., 1991; Stewart et al., 1970) give evidence that 1.5 μg/day of dietary B₁₂ is not sufficient to maintain hematological status and serum B₁₂ in half of the subjects studied. The meager data provided by the studies of vegetarians indicate that the B₁₂ average requirement should probably be at least 1.5 μg/day, but a higher average requirement is not ruled out.

Supportive Data: Maintenance of B₁₂ Body Stores

Various studies have indicated losses of 0.1 to 0.2 percent/day of the B₁₂ pool (e.g., Amin et al., 1980; Boddy and Adams, 1972; Heyssel et al., 1966; Reizenstein et al., 1966) regardless of the size of the pool. A loss of 0.2 percent appears to be typical for individuals who do not reabsorb biliary B₁₂ because of pernicious anemia (Boddy and Adams, 1972). A person with a B₁₂ pool of 1,000 μg and a loss of 0.1 percent would excrete 1 μg of B₁₂ daily, and a person with a 3,000-μg pool would excrete 3 μg daily. If only 50 percent of dietary B₁₂ is absorbed, the amounts required daily to replenish the pools are 2 and 6 μg of B₁₂, respectively. The higher value would lead to less efficient use of B₁₂, but the larger store of B₁₂ would cover a longer period of inadequate B₁₂ intake or absorption.

With a 0.1 percent loss, the period of protection afforded by the B₁₂ pool can be estimated if the lowest pool size consistent with health is also known. If it is assumed that this value is 300 μg (derived from Bozian and coworkers [1963]), there is no absorption of B₁₂ from food or supplements, and the enterohepatic circulation is intact, then stores of 1 mg would be expected to meet the body's needs for 3 years, 2 mg for about 5 years, and 3 mg for about 6 years. A 1.5 percent loss would reduce these estimates to 2, 3.6, and 4 years (see Appendix N for the method used to obtain these values).

The extent of the supply of reserve B₁₂ may be an important consideration when persons approach the age of 50 and the risk increases for food-bound B₁₂ malabsorption secondary to atrophic gastritis (see “Factors Affecting the Vitamin B₁₂ Requirement” and section “Adults Ages 51 Years and Older”). Because the absorption of B₁₂ from fortified foods, oral supplements, or the bile does not appear to be impaired, the combination of stores and absorbed crystalline B₁₂ may cover needs for an extended period.

The estimates above for the period of protection afforded by body stores are consistent with the periods required to develop overt signs of B₁₂ deficiency after a total gastrectomy; for example, megaloblastic anemia has been typically diagnosed 2 to 5 years after a total gastrectomy (Chanarin, 1990).

Possible Ancillary Method: Maintenance of a Serum B₁₂ Concentration That Is Consistent with a Normal Circulating MMA Value

Several investigators have urged the use of the serum MMA concentration as the most sensitive indicator of B₁₂ status (Lindenbaum et al., 1990; Moelby et al., 1990; Savage et al., 1994b; Stabler et al., 1996). This indicator could not be used as the criterion for setting the EAR for B₁₂ because of a lack of direct data. At least one study (Lindenbaum et al., 1994) relates serum B₁₂ to circulating MMA values. None link MMA with B₁₂ intake. Moreover, although MMA is a metabolite that accumulates abnormally when the B₁₂ supply is low, studies have not yet convincingly demonstrated that elevated MMA caused by insufficient B₁₂ intake has adverse health consequences. However, because MMA values hold promise as a criterion for estimating the B₁₂ requirement in the future, an indirect approach was used to estimate a requirement for B₁₂ as a means of confirming or refining the EAR value derived by using the primary approach. For example, because the serum B₁₂ value of 150 pmol/L (200 pg/mL) appears to be the level at which half the population would have an elevated MMA value (Lindenbaum et al., 1994), one could select the dietary intake that would maintain this value in healthy individuals in that population.

In a study of 548 surviving members of the original Framingham Heart Study cohort, aged 67 to 96 years, and 117 healthy control subjects younger than 65 years, Lindenbaum and colleagues (1994) reported on serum B₁₂, MMA, and homocysteine values (Table 9-8). These investigators used a cutoff value equal to or greater than 260 pmol/L (350 pg/mL) of B₁₂ as adequate; more than 15 percent of subjects below the cutoff value had elevated MMA concentrations whereas fewer than 10 percent of subjects above the cutoff did. Serum creatinine was elevated in 10 of those with both increased MMA and low B₁₂ values, which would indicate confounding abnormal renal function. Slightly more than 40 percent of the 70 elderly subjects with serum B₁₂ less than 150 pmol/L (200 pg/mL) had an elevated MMA concentration.

TABLE 9-8

Vitamin B₁₂ Status: Occurrence of Low Serum Values for Two Age Groups.

Studies of B₁₂ intake and serum B₁₂ concentration provide very limited information on the relationship of the two. In Finland, vegans consuming an uncooked (“living food”) diet were estimated to consume a mean of 1.8 μg/day of B₁₂ (range 0 to 12.8 μg) (Rauma et al., 1995), but the accuracy of the dietary intake data is uncertain. The 16 vegans who ate seaweed (the main source of B₁₂ reported) had B₁₂ concentrations twice as high as those not eating seaweed (mean of 220 pmol/L [300 pg/mL] compared with 105 pmol/L [142 pg/mL]). On this diet 57 percent of the vegans had serum B₁₂ concentrations less than 200 pmol/L (270 pg/mL). A study by Draper and colleagues (1993) provided dietary data on vegans that were not sufficient for drawing conclusions about diet-B₁₂ relationships. Neither Garry and coworkers (1984) nor Sahyoun and colleagues (1988) separated data with regard to supplement use, so their data are not interpretable for setting EARs. A study of a macrobiotic population (Miller et al., 1991) revealed that more than half of the adults had low serum B₁₂ concentrations and nearly one-third were excreting high amounts of MMA, but dietary information from the study was not sufficient for drawing conclusions. Moreover, studies need to be conducted in younger persons in whom B₁₂ absorption is more likely to be normal.

B₁₂ EAR and RDA Summary, Ages 19 through 50 Years

On the basis of hematological evidence and serum B₁₂ values, the EAR for B₁₂ is estimated to be 2 μg/day for men and women ages 19 through 50 years. Sufficient data were not available to enable differences in requirements to be discerned for men and women in these age groups.

The RDA for B₁₂ is set by assuming a coefficient of variation (CV) of 10 percent (see Chapter 1) because information is not available on the standard deviation of the requirement for B₁₂; the RDA is defined as equal to the EAR plus twice the CV to cover the needs of 97 to 98 percent of the individuals in the group (therefore, for B₁₂ the RDA is 120 percent of the EAR).

Evidence Considered in Estimating the Average Requirement

Because 10 to 30 percent of people older than 50 years are estimated to have atrophic gastritis with low stomach acid secretion (Andrews et al., 1967; Hurwitz et al, 1997; Johnsen et al., 1991; Krasinski et al., 1986), they may have decreased bioavailability of B₁₂ from food. Therefore, because of the high prevalence of this condition, 50 percent bioavailability of dietary B₁₂ (see Box 9-2) cannot be assumed for this age group, and the EAR would be higher than 2.0 μg. Similarly, 2.4 μg of B₁₂, which is the RDA for younger adults, might not meet the needs of 97 percent of this large age group. There is not sufficient information on which to base a bioavailability correction factor for persons with atrophic gastritis who obtain their B₁₂ from animal foods. However, because the bioavailability of crystalline B₁₂ is not altered in people with atrophic gastritis, the same EAR and RDA would apply if the dietary sources of B₁₂ were foods fortified with B₁₂, supplements, or a combination of both.

B₁₂ EAR and RDA Summary, Ages 51 Years and Older

The EAR and RDA for B₁₂ for adults ages 51 years and older are the same as for younger adults but with the recommendation that B₁₂-fortified foods (such as fortified ready-to-eat cereals) or B₁₂-containing supplements be used to meet much of the requirement.

The RDA for B₁₂ is set by assuming a coefficient of variation (CV) of 10 percent (see Chapter 1) because information is not available on the standard deviation of the requirement for B₁₂; the RDA is defined as equal to the EAR plus twice the CV to cover the needs of 97 to 98 percent of the individuals in the group (therefore, for B₁₂ the RDA is 120 percent of the EAR).

Evidence Considered in Estimating the Average Requirement

Absorption and Utilization of B₁₂. There is some evidence that the absorption of B₁₂ may increase during pregnancy. An increase in the number of intrinsic factor–B₁₂ receptors was observed in pregnant mice and found to be regulated by placental lactogen (Robertson and Gallagher, 1983). A greater absorption of oral B₁₂ was reported from the single study of pregnant women (Hellegers et al., 1957), but the methods used do not permit quantification of the increase.

Serum total B₁₂ concentrations begin to decline early in the first trimester. In a longitudinal Dutch study of 23 subjects, serum B₁₂ fell significantly by the end of the first trimester, more than could be accounted for by hemodilution (Fernandes-Costa and Metz, 1982). There were further decreases through the sixth month to about half of nonpregnancy concentrations. Some of the later decrease was due to hemodilution. However, transcobalamin I and III increase during the second and third trimesters, and transcobalamin II increases sharply in the third trimester to about one-third more than in nonpregnant, nonlactating control subjects (Fernandes-Costa and Metz, 1982).

Transfer to the Fetus. The serum B₁₂ concentration of the newborn is twice that of the mother, decreasing to adult concentrations at about 6 to 7 months postpartum (Luhby et al., 1958). The placenta concentrates B₁₂, which is then transferred to the fetus down a concentration gradient. Fetal and maternal B₁₂ serum concentrations are quite strongly correlated (Fréry et al., 1992). It appears that only newly absorbed B₁₂ is readily transported across the placenta and that maternal liver stores are a less important source of the vitamin for the fetus (Luhby et al., 1958). This implies that current maternal intake and absorption of the vitamin during pregnancy have a more important influence on the B₁₂ status of the infant than do maternal B₁₂ stores. The importance of adequate maternal intake during pregnancy is supported by the appearance of B₁₂ deficiency in infants at 4 to 6 months when their mothers have been strict vegetarians for only 3 years (Specker et al., 1990).

Fetal Accumulation. The human fetus accumulates an average of 0.07 to 0.14 nmol/day (0.1 to 0.2 μg/day) of B₁₂, a range based on three studies of the liver content of infants born to women who were adequate in B₁₂ (Baker et al., 1962; Loria et al., 1977; Vaz Pinto et al., 1975) and an assumption that the liver contains half the total body B₁₂ content. Placental B₁₂ is negligible (0.01 nmol/L [14 ng/L]) (Muir and Landon, 1985). The low body content of B₁₂ in the newborn implies that pregnancy is unlikely to deplete maternal stores.

B₁₂ EAR and RDA Summary, Pregnancy

On the basis of a fetal deposition of 0.1 to 0.2 μg/day throughout pregnancy and evidence that maternal absorption of the vitamin becomes more efficient during pregnancy, the EAR is increased by 0.2 μg/day during pregnancy. No distinction is made for the age of the mother.

The RDA for B₁₂ is set by assuming a coefficient of variation (CV) of 10 percent (see Chapter 1) because information is not available on the standard deviation of the requirement for B₁₂; the RDA is defined as equal to the EAR plus twice the CV to cover the needs of 97 to 98 percent of the individuals in the group (therefore, for B₁₂ the RDA is 120 percent of the EAR).

Evidence Considered in Estimating the Average Requirement

As described earlier, the average amount of B₁₂ secreted in the milk of mothers with adequate B₁₂ status is approximately 0.33 μg/day during the first 6 months of lactation. During the second 6 months, the average amount of B₁₂ secreted is slightly less: 0.25 μg/day.

The concentration of B₁₂ in milk is usually similar to that in maternal plasma. In some studies, human milk and maternal plasma concentrations are strongly correlated (Srikantia and Reddy, 1967) but in others they are not (Casterline et al., 1997; Donangelo et al., 1989). The correlation appears to be stronger when maternal B₁₂ status is marginal (Fréry et al., 1992).

Current maternal intake of the vitamin may have an important influence on secretion of the vitamin in milk. In several studies of infants with clinical signs of B₁₂ deficiency caused by low maternal intake or absorption of the vitamin, maternal plasma concentrations of the vitamin were found to be normal or low normal, suggesting that maternal B₁₂ stores are less important than current maternal intake (Hoey et al., 1982; Johnson and Roloff, 1982; Kuhne et al., 1991; Sklar, 1986). This is also indicated by the observation that the length of time that mothers had been strict vegetarians was not correlated with the urinary MMA concentrations of their infants (Specker et al., 1988).

Low B₁₂ concentrations in human milk occur commonly in two situations involving inadequate intake: when the mother is a strict vegetarian and in developing countries where the usual consumption of animal products is low. When the B₁₂ status of the mother is marginal, further maternal depletion may occur as reflected in decreasing concentrations of maternal plasma B₁₂ (Black et al., 1994; Shapiro et al., 1965).

B₁₂ EAR and RDA Summary, Lactation

To estimate the EAR for lactation, 0.33 μg/day of B₁₂ is added to the EAR of 2 μg/day for adolescent girls and adult women; the result is rounded up.

The RDA for B₁₂ is set by assuming a coefficient of variation (CV) of 10 percent (see Chapter 1) because information is not available on the standard deviation of the requirement for B₁₂; the RDA is defined as equal to the EAR plus twice the CV to cover the needs of 97 to 98 percent of the individuals in the group (therefore, for B₁₂ the RDA is 120 percent of the EAR).

Adverse Effects

No adverse effects have been associated with excess B₁₂ intake from food or supplements in healthy individuals. There is very weak evidence from animal studies suggesting that B₁₂ intake enhances the carcinogenesis of certain chemicals (Day et al., 1950; Georgadze, 1960; Kalnev et al., 1977; Ostryanina, 1971). These findings are contradicted by evidence that increased B₁₂ intake inhibits tumor induction in the human liver, colon, and esophagus (Rogers, 1975). Some studies suggest a possible association between high-dose, parenterally administered B₁₂ (0.5 to 5 mg) and acne formation (Berlin et al., 1969; Dugois et al., 1969; Dupre et al., 1979; Puissant et al., 1967; Sherertz, 1991). However, the acne lesions were primarily associated with hydroxocobalamin rather than cyanocobalamin, the form used in the United States and Canada. Furthermore, iodine particles in commercial B₁₂ preparations may have been responsible for the acne. In conclusion, the evidence from these data was considered not sufficient for deriving a Tolerable Upper Intake Level (UL).

Studies involving periodic parenteral administration of B₁₂ (1 to 5 mg) to patients with pernicious anemia provide supportive evidence for the lack of adverse effects at high doses (Boddy and Adams, 1968; Mangiarotti et al., 1986; Martin et al., 1992). Periodic doses of 1 mg are used in standard clinical practice to treat patients with pernicious anemia. As indicated earlier, when high doses are given orally (see “Absorption”) only a small percentage of B₁₂ can be absorbed from the gastrointestinal tract, which may explain the apparent low toxicity.

Special Considerations

B₁₂-deficient individuals who are at risk for Leber's optic atrophy should not be given cyanocobalamin to treat the B₁₂ deficiency. Leber's optic atrophy is a genetic disorder caused by chronic cyanide intoxication (present in tobacco smoke, alcohol, and some plants). Reduced serum B₁₂ concentrations have been associated with a reduced ability to detoxify the cyanide in exposed individuals (Foulds, 1968, 1969a, b, 1970; Wilson and Matthews, 1966). Cyanocobalamin may increase the risk of irreversible neurological damage (from the optic atrophy). Hydroxocobalamin is a cyanide antagonist and therefore not associated with adverse effects when given to these individuals.