Non-protein nitrogen constituents in mature human milk and colostrum

The significance of the non-protein nitrogen (NPN) fraction of human milk (HM) in the nutrition of infants has received little attention. NPN constitutes 25% of the total nitrogen in mature milk (MM) and 28% in colostrum. NPN was determined by Kjeldahl analysis of the TCA soluble fraction of HM, dialysates of HM, fractions from gel filtration and ultrafiltrates of HM. NPN determination by TCA precipitation was found to be identical to gel filtration (25%) and a superior method for determining NPN, compared to dialysis or ultrafiltration which underestimates the NPN fraction, possibly due to Donnan effects. Using specific assays, the NPN fraction of MM and colostrum was found to contain the following compounds: urea (54%, 16%), uric acid (.6%, .4%), ammonia (0.4%, 0.13%), and MM: creatinine (1.84%), creatine (2.6%), free AA (7.4%), choline (2.7%), and glucosamine (3.9%). These compounds account for ~73% of the NPN in MM, leaving 27% unaccounted for. To further study and isolate the NPN constituents, whey samples prepared by ultracentrifugation were fractionated by Sephadex G-25 gel filtration. Three fractions were eluted containing 70, 16, and 12% of the whey nitrogen. The first fraction comprises whey protein (MW>10,000); fractions two and three were further separated by gel filtration on Sephadex G-15. The first Sephadex G-15 fraction contained peptides of intermediate MW. Futher study of these fractions will help identify and quantify the unknown constituents of the NPN in HM.