Human milk protein was determined by three colorimetric methods and by Kjeldahl analysis. The distribution of nitrogen (N) and protein was determined within various milk compartments. Total N, whey, casein, nonprotein nitrogen (NPN), cell N and N in the fat fraction were analyzed by micro-Kjeldahl analysis after a series of centrifugation and ultracentrifugation separations. Fresh milk samples (colostrum, transitional milk and mature milk) were centrifuged at 500 X g to separate milk cells and at 5000 X g to skim the milk. Decelled milk and skimmed milk were ultracentrifuged at 189,000 X g to separate fat and casein micelles from whey. NPN was analyzed after trichloroacetic acid precipitation. Whole milk, decelled milk, skimmed milk and whey were analyzed for protein with the Lowry method, modified for fat-containing samples, the Bradford dye-binding assay (Bio-Rad) and the Pierce bicinchoninic acid (BCA) assay. Cell nitrogen had a tendency to be lower in mature milk than in colostrum. Colostrum contained only 6% casein protein, whereas mature milk contained 13%. Fat from skimming was lower in N than fat from ultracentrifugation. Average NPN levels were similar for milk from all three lactation periods, and constituted 10% of colostrum N and 25% of mature milk N. Protein determined by the Bio-Rad method on whole milk samples had the lowest variability (square root MSE) when correlated to Kjeldahl values. All three assays had lower variability when analyzing whey and skimmed milk than when analyzing whole milk. The Lowry method and the Bio-Rad method had low variability for whey and skimmed milk samples, but the Lowry method yielded analytical values closest to Kjeldahl protein values. The BCA method consistently overestimated Kjeldahl protein by 30%.
The metal-binding property of alpha-lactalbumin (alpha-LA) in human milk was studied and compared to that of bovine milk alpha-LA. Gel filtration on Sephadex G-75 at physiological pH and ionic strength separated alpha-LA in human and bovine milk from most other proteins. The only metal ion associated with alpha-LA under these conditions was Ca2+. Minor protein contaminants were removed by ion-exchange chromatography, and the ratio of Ca2+:alpha-LA was determined in the isolated protein preparations. Concentrations of alpha-LA in mature human milk were between 1.03 and 1.57 mg/ml; the Ca2+ concentration bound to alpha-LA varied, yielding a molar ratio of Ca2+:alpha-LA of approximately 1:1 (0.82-1.41 mol Ca2+/mol alpha-LA) in mature milk. Gel filtration with excess Ca2+ in the running buffer showed that there is another weaker binding site for Ca2+, but this binding does not occur under physiological conditions. Less Ca2+ was bound to bovine alpha-LA (0.6-0.9 mol Ca2+/mol alpha-LA) than to human alpha-LA. Calcium binding was abolished at pH 3.0 and resulted in a substantial increase in the hydrodynamic radius of alpha-LA. Reconstitution of human alpha-LA with Ca2+ and other divalent cations at native pH (6.8) and ionic strength showed a binding specific for Ca2+. Since only 1% of calcium from human milk and 0.15% from bovine milk is alpha-LA bound, alpha-LA is probably unimportant with respect to calcium nutrition of the infant. However, the metal binding of alpha-LA may have a biological significance through its role in the lactose synthase complex.
Little is known about the absorption of magnesium from infant diets. Magnesium bioavailability was evaluated from infant diets that varied by protein and carbohydrate source; magnesium, calcium, and phosphorus contents; and the form of magnesium fortification. Diets were separated into soluble, insoluble, and fat fractions to determine magnesium distribution. Most of the magnesium (> 62%) was found in the soluble fraction. Gel filtration of the soluble fraction from all diets studied showed that > 95% of magnesium is free or associated with low-molecular-weight compounds. Distribution of 28Mg and native magnesium in fractions of the diets was similar, thus validating the use of an extrinsic label. In vitro digestion decreased the percent insoluble magnesium from as high as 35% to 2-8%. Whole-body retention of 28Mg-labeled diets in suckling rat pups 4 h after oral intubation ranged from 51% to 92%. No significant differences were found between human milk, cow milk, and infant formula. In conclusion, magnesium from the infant diets studied has high bioavailability, and moderate differences in their composition do not affect bioavailability significantly.
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