Subjects typically arrived between 8:00–10:00 am, approximately 1 ½ hours after the last feeding. This was done to standardize milk collection and reduce potential diurnal variation in milk composition. Every attempt was made to ensure mothers were fasted at least 2 hours; however, it was not always confirmed. Specific human breast-milk analytes were measured in milk collected from one full breast expression. At collection, the entire contents of one breast at 1-month (mean volume 2.4 ± 0.2 oz.) were collected using an electric breast pump (Medela, Inc.) ensuring the collection of fore-, mid-, and hind-milk within each sample.
Thoroughly mixed human breast-milk was divided into ten aliquots. All aliquots were stored at −80°C until analyses. Prior to analyses, aliquots were thawed on ice and milk fat was separated from the aqueous phase by centrifugation at 3,000 × g for 10 minutes [30 (link)–32 (link)]. The resulting skimmed milk was assayed using commercially available immunoassay kits for insulin, leptin, IL-6 and TNF-α. Glucose was measured by the glucose oxidase method (2300 STAT Plus, Yellow Springs Instruments).
Thoroughly mixed human breast-milk was divided into ten aliquots. All aliquots were stored at −80°C until analyses. Prior to analyses, aliquots were thawed on ice and milk fat was separated from the aqueous phase by centrifugation at 3,000 × g for 10 minutes [30 (link)–32 (link)]. The resulting skimmed milk was assayed using commercially available immunoassay kits for insulin, leptin, IL-6 and TNF-α. Glucose was measured by the glucose oxidase method (2300 STAT Plus, Yellow Springs Instruments).
