Effect of Breast Milk Ingestion Upon the Thyroxinemia of Suckling Rat Pups
The purpose of our studies was to develop a method for separation of milk iodothyronines in order to define the thyroid hormone metabolites present both in expressed milk from lactating dams and the stomach contents of their sucklings. Breast milk from in vivo radioiodine-labelled and -unlabelled lactating dams was subjected to chloroform: methanol extraction followed by CaCl2 precipitation; the lyophilized extract was then further purified using a Bio-Rad ion-exchange resin column sequentially eluted with increasing concentrations of acetic acid. The iodothyronine eluate was then evaporated under nitrogen and a reconstituted aliquot injected onto a DuPont C-8 column and absorbance measured at 300 nm. Extracts of dams’ milk contained only 8–10% of a Na 125I cpm initially present in the whole milk. Recovery was dramatically increased by enzymatic digestion using either pepsin, trypsin, or pancreatin. Of these enzymes, we chose to investigate further the activity of pepsin, which most effectively liberated the iodothyronines of interest. Five litters of 12- and 14-day old suckling rats were separated from their dams for 6 hrs, after which time one animal per litter was sacrificed (“pre-suckling” animals). The remaining pups were permitted to suckle for 90 min; at 0, 1, 2, 3, and 4 hrs post-suckling, one animal per litter was sacrificed, trunk blood collected, and the stomachs removed. The latter were homogenized and then extracted as described above.