Bioactive constituents. Enzymes and other bioactive constituents of human milk may alter the composition of expressed milk (Greenberg and Graves (1984), even at temperatures well below 0° C. (Berkow et al., 1984; Bitman et al., 1983).
Bound forms. Several of the vitamins (such as vitamin D, folate, and pantothenic acid) are secreted bound to other compounds, and they must be released before they can be completely extracted or detected. For example, accurate measurement of the total content of pantothenic acid in human milk requires double enzyme hydrolysis (Song et al., 1984).
Distribution in aqueous and lipid fractions. Vitamin D and its metabolites are secreted in the aqueous fraction of human milk and are attached to binding proteins (Hollis et al., 1982), but on standing they diffuse to the lipid fraction of milk. Thus, whether aqueous or lipid solvents are used should be determined by the handling procedure.
Other sources of measurement errors. Commercial sources of reagents such as enzymes may be contaminated with vitamins and be responsible for falsely elevated levels in milk (Song et al., 1984). Many of the water-soluble vitamins are measured by microbiological assays. Care must be taken to ensure that the vitamin to be measured is stable under the extraction method employed and that the vitamin is converted to a form that can be utilized by the test organism. For example, the folate content of human milk is likely to be underestimated unless an antioxidant is used to prevent it from being oxidized, conjugase pretreatment is performed to cleave the long-chain forms of the vitamin, heat treatment is applied to release the folate from its binding proteins before microbiological analysis, and test organisms are selected that are able to use all the forms of folate in the samples (O'Connor et al., 1990a).
The reproducibility and validity of techniques used in different studies could not always be ascertained by the subcommittee. Thus, the data on the nutrient content of human milk must be interpreted with caution. Large variations reported for many milk constituents may reflect improper sampling or analytic inaccuracies or both rather than true biologic variance.
In addition to the methodologic concerns just described, there are problems of measurement and detection specific to nonnutrient constituents, as follows:
The leukocytes in human milk are difficult to identify because their morphology is altered by the presence of many intracytoplasmic lipid bodies.
Certain constituents, such as secretory immunoglobulin A (IgA), exist in a different physical form than they do in other tissues, such as blood, and therefore require discrete detection procedures.
The titer of specific antibodies in human milk depends on whether the woman has recently been exposed to the relevant immunogen via the intestinal or respiratory tract.