Composition and ultrastructure of calcium phosphate-citrate complexes in bovine milk systems.

The present studies show that the colloidal calcium phosphate of cows' milk has a (Ca + Mg)/Pi ratio of 1.67 (+/- 0.10; n = 22) and contains citrate, Mg and Zn at molar ratios to Ca averaging 0.05, 0.03 and 0.003, respectively. The composition of the natural colloidal phosphate of milk is similar to the precipitates formed by neutralization of ultrafiltrates obtained from acidified milks, and to that of the calcium phosphate-enriched fraction produced by extensive enzymic hydrolysis of the casein micelles in milk. Examination by electron microscopy of these artificial preparations of milk calcium phosphate revealed in both a very fine and uniform substructure which consisted of granules having an average, true diameter of approx. 2.5 nm. The size and shape of these tiny granules closely resemble the morphologies reported for the colloidal phosphate particles in native casein micelles, as well as for the subunits of amorphous calcium phosphate observed during calcification in other biological systems such as mitochondria and bone.

Amorphous calcium phosphate in casein micelles of bovine milk.

The calcium phosphate remaining after hydrazine deproteination of casein micelles isolated from bulk skim milk exhibits under the electron microscope a very fine and uniform granularity being formed by small subunits with a true diameter of approximately 2.5 nm. This material, which is about 10 percent by weight citrate, termed calcium phosphate citrate (CPC) complex, also contains Mg and Zn at molar ratios of 0.03 and 0.003 respectively. Radial distribution function (RDF) and infrared analyses show that CPC is a Mg-containing amorphous calcium phosphate (ACP) similar to synthetic and cytoplasmic ACP. presence of CPC in casein micelles as an amorphous colloid bonded with phosphoproteins provides the means for storing in milk large amounts of Ca (16 mM) and Pi (10 mM) in a readily utilizable form but at a higher ion concentration than found in biological solutions.

Composition and size distribution of bovine casein micelles.

Chromatography of glutaraldehyde-fixed skim-milk on controlled-pore glass (CPG-10, 300 nm) gave three micellar fractions whose averaged diameters, measured by electron microscopy, decreased progressively with increasing elution volume. Casein micelles with diameters up to 680 nm were detected. The casein composition of the same fractions from unfixed skim-milk was determined. As the fraction elution volume increased, kappa-casein varied from 7.7 to 11.4% of total casein, giving alpha s/kappa ratios of 6.1, 4.7 and 3.3. A plot of kappa-casein content versus micelle surface-to-volume ratio for skim-milk and the column fractions approximated to a straight line. Re-calculation of the published results from two other studies also gave linear relationships between kappa-casein content and surface area for artificial micelles. The three regression lines thus obtained had small intercepts. It was concluded that the data indicated the same fundamental structure for casein micelles, with a predominant surface location for kappa-casein, whether the micelles are natural or artificial and whether they are aggregated by Ca2+ alone or by Ca2+ together with calcium phosphate-citrate complex.

Levels and location of adenosine 5'-triphosphate in bovine milk.

Bovine milk systems were analysed for adenosine 5'-triphosphate (ATP) using the luciferase-ATP reaction in a liquid scintillation counter. Approximately 0.2 mumol ATP/l milk serum were evident both in whole milks and the corresponding skim-milks. ATP was not detectable in skim-milk ultrafiltrates. These findings indicated that ATP was present in a non-dialysable portion of skim-milk. Centrifugation of whole milks from individual cows at 5500 g for 15 min at 10 degrees C yielded skim-milks essentially devoid of somatic cells and bacteria. However, the ATP in the skim-milks decreased by less than 20% compared with the whole milks indicating that the calcium phosphate-citrate (CPC)--caseinate micelles were the source of the ATP. ATP was not detectable in colloidal phosphate-free milk, from which CPC had been removed, confirming that the ATP was sequestered in the constituent CPC. Likewise, the occurrence of significant amounts of Mg, another potent stabilizer of amorphous calcium phosphate (ACP) in other biological systems, was confirmed in the colloidal phosphate of milk. From 0.13 to 0.31 mumole ATP/1 (mean 0.23) was found in the 9 milk samples studied. The discovery of small but appreciable levels of ATP in the CPC of milk provides further evidence for the analogy previously shown to exist between the CPC complex of milk and the ACP which accumulates in mitochondria. The latter has been postulated to provide an essential precursor for crystalline bone salts to form in ordered calcification processes. The implications of these findings in the biosynthesis of milk are briefly discussed.

Developments in column chromatography for the separation and characterization of casein micelles.

Skim-milk was fractionated by permeation chromatography on CPG-10 (300 nm) at 20 degrees C with synthetic milk serum. The elution profile, which was highly reproducible, showed 3 partly resolved peaks. On treatment with 0.1% glutaraldehyde before chromatography, a similar profile was obtained indicating no appreciable micellar dissociation during chromatography of the milk. The casein composition of the eluent fractions, determined by quantitative chromatography on hydroxyapatite, showed that 12.1% of total casein was associated with the low molecular weight peak. The micelle content of beta- and kappa-casein increased with increasing elution volume, and the alphas/kappa ratio showed a progressive diminution from 4.3 to 2.3. Large differences were found in the colloidal phosphate contents and the weight average diameters of the micelle fractions which clearly show that CPG-10 fractionates on the basis of molecular size.