Laboratory methods.
Hemoglobin was measured using a CBC5 Coulter Counter or cyanmethemoglobin method using a Colorimeter (Corning). Serum retinol and vitamin E were measured using HPLC (15). Stool specimens were examined macroscopically for helminths and microscopically for ova and cysts. Active syphilis was defined as positive results for sera antibodies in both the VDLR (Murex Diagnostic) and Treponema pallidum hemagglutination (Fujirebio) tests. Vaginal and cervical swabs were examined microscopically for infections caused by Trichomonas vaginalis. The presence of malaria parasites was ascertained from Giemsa-stained thick and thin smear blood films. CD4+ T-cell counts were determined with the use of FACSCount and FACSCan systems (Becton Dickinson).
Immediately after breast milk collection, samples were placed on ice and transported to the Microbiology and Immunology Laboratory at Muhimbili National Hospital. On the same day, 1 aliquot was centrifuged at 1500 × g for 12 min at 4°C. The cell-free aqueous milk fraction and milk pellet were separately stored at −70°C until laboratory testing at Children’s Hospital Boston. Flame photometry was used to analyze Na and K concentrations in the aqueous fraction by using the Instrumentation Laboratory 943 Flame Photometer (Instrumentation Laboratory) (16). Each sample was atomized and mixed with propane gas and sprayed into a chimney and ignited. Na produces a characteristic flame with an emission wavelength read at 589 nm; K was read at 776 nm. The day-to-day variations of Na at 78.4 and 14.9 mmol and K at 28.1 and 5.4 mmol were 1.0, 1.3, 1.1, and 1.6%, respectively.