ods 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.

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