Casein content of human 1,2

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1 Original Research Communications-methods Casein content of human 1,2 Bo L#{246}nnerdal and Elisabet Forsum Introduction ABSTRACT Three methods for estimating the casein content of human milk were tested; isoelectric precipitation with washing and correction for co-precipitating proteins, sedimentation by ultracentrifugation, and indirect analysis (ie analyzing for the content of the major whey proteins and subtracting these from the total protein content). Gel electrophoresis and amino acid analysis were used to confirm some of the results. The casein content (mg/ml) of mature human milk (n = 9) was 2.33 ± 1.69 by isoelectric precipitation, 1.80 ± 0.48 by sedimentation and 2.96 ± 1.08 by the indirect approach. A probable partition of nitrogen in breast milk would be casein N:whey protein N:non-protein N of 20:50:30; ie the correct ratio of casein nitrogen:whey nitrogen is -.20:80. Analysis of trace elements and minerals demonstrates that of total Ca 10%, Mg 5%, Zn 28%, Cu 17%, and Fe 27% is bound to casein when prepared by ultracentrifugation while isoelectric precipitation causes a redistribution of some of these elements. Since the protein ratio of human milk is considered a guideline when manufacturing infant formulas, these findings should be considered with regard to infant nutrition. Am J Clin Nuir 1985;l KEY WORDS Human milk, casein, whey, whey proteins, iron, copper, zinc, calcium, magnesium, trace elements, minerals The traditional method for classifying milk proteins was developed by Rowland (1) and intended primarily for bovine milk proteins. According to this approach, casein is precipitated at its isoelectric point, ph 4.6. Proteins remaining in solution are called whey proteins. This procedure has also been applied to human milk, but it was recognized early that human casein differed from cows casein in its physico-chemical properties, ie, the precipitate formed by human casein at ph 4.6 was looser and softer than that formed by bovine casein. Casein as a class of proteins consists of several subunits (a, and sc-casein) which form micelles with Ca and P04 giving milk its characteristic white appearance. There are differences between the caseins from different species, however, eg the micelles in human milk are considerably smaller than those of bovine milk (2). Also, the presence of a-casein in human milk has not been clearly demonstrated. No method has been developed to satisfactorily quantitate the content of casein in human milk. Using isoelectric precipitation human casein nitrogen has been estimated to comprise about 35% of total nitrogen in human milk (3). A figure of4o% has been widely accepted and is used as a guideline by infant formula manufacturers when cow s milk protein content is modified for infant feeding. This modification involves adjustment of the ratio of casein nitrogen to whey protein nitrogen in cow s milk (80:20) to mimic the ratio found I From the Department of Nutrition, University of California, Davis, CA and the Department of Medical Nutrition, Huddinge University Hospital, Karolinska Institute, Huddinge, Sweden. 2 Address reprint requests to: Bo L#{246}nnerdal, Department of Nutrition, University of California, Davis, CA Received April 13, Accepted for publication July 24, The American Journal ofciinical Nutrition 41: JANUARY 1985, pp Printed in USA 1985 American Society for Clinical Nutrition

2 114 LONNERDAL AND FORSUM in human milk (40:60). In this way, a more balanced amino acid profile of the formula is obtained. Such modifications may also affect the bioavailability of trace minerals. For example, Lonnerdal et al (4) presented data indicating the bioavailability of zinc is lower from a formula with a casein N:whey N ratio of 80:20 than from a formula with a corresponding ratio of 40:60 suggesting that zinc in cow s milk may be less available when bound to casein than when present in the whey fraction of milk. Modifications in the ratio of casein to whey proteins of cow s milk should thus be evaluated for the nutritional consequences to the formula-fed infant. Since the casein:whey ratio may affect trace element bioavailability it becomes interesting to know the mineral distribution in human milk as a guideline when the optimal ratio between casein and whey in formulas is considered. In an earlier study (5) it was found that over a ph-range of similar amounts of protein-nitrogen precipitated from human milk; significant amounts of this precipitate were non-casein proteins, eg, lactoferrin. Also, by an indirect approach (ie by estimating the total amount of nitrogen in breast milk, nonprotein nitrogen and the contribution of nitrogen from individual whey proteins) casein nitrogen was calculated to be considerably lower than 40% of breast milk nitrogen. In this study the partition of nitrogen between total nitrogen, non-protein nitrogen, protein nitrogen and individual whey proteins has been investigated in nine human milk sampies and the amount of nitrogen equivalent to the theoretically maximal content of casein nitrogen has been calculated. Also studied were the amounts of nitrogen precipitated at ph 4.6 and of nitrogen-containing material sedimented by ultracentrifugation. Furthermore, the amounts of calcium, magnesium, zinc, copper and iron recovered in casein are reported as prepared by the ultracentrifugation and isoelectric precipitation methods. Materials and methods Milk samples. Samples were obtained fresh from healthy, exclusively breast-feeding Swedish donors after 4-16 weeks of lactation. The protocol was approved by the Human Subjects Committee at University of Uppsala. The milk was collected at the mid-morning meal (8-10 AM) and was expressed by a manual breast pump (Gentle Flow, Eklund and Co. Davis, CA). All parts of the pump in contact with the milk and the plastic storage bottles had been thoroughly washed, soaked in 10% nitric acid and rinsed with distilled deionized water. All samples were defatted by centrifugation at 4,000 g for 30 mm at 4#{176}C. Isoelectric precipitation. Milk samples (20 ml) were adjusted to ph 4.6 by slow addition of hydrochloric acid (1 M) during stirring at 20#{176}C. After 1 h, the samples were centrifuged at 10,000 g for 15 mm at 20#{176}C. Supernatants were decanted and the pellets suspended in saline (5 ml) and adjusted to ph 4.6 with hydrochloric acid. Following 1 5 mm of stirring, the suspended pellets were centrifuged as above. The resulting supernatants were combined with the previous supernatants and the pellets (isoelectric precipitates) were dissolved in saline (5 ml) adjusted to ph 8.0 with sodium hydroxide. Sedimentation. Milk samples (1 5 ml) were ultracentrifuged (MSE superspeed 75 Measuring & Scientific Equipment Ltd., London) at 105,000 g for 30 mm at 20#{176}C. Supernatants were decanted and the sediments were dissolved in saline (5 ml) adjusted to ph 8.0 with sodium hydroxide. Protein nitrogen (PN) and non-protein nitrogen (NPN). Estimations were as follows: 2 ml 24% trichloroacetic acid was added to 2 ml milk and the samples were centrifuged at 27,000 g for 60 mm after which the supernatants were decanted and their nitrogen content analyzed (NPN). The precipitates were dissolved in 0. 1 M NaOH and analyzed for nitrogen (PN). Nitrogen analysis. Analysis was performed by a modified micro-kjeldahl method described earlier (6). Milk samples and dissolved precipitates and sediments were analyzed for total nitrogen. For conversion of nitrogen to protein the factor of 6.25 was used. Individual proteins. Lactoferrin was determined by immunoelectrophoresis according to Laurell (7). Serum albumin, ct-lactalbumin and IgA were determined by radial immu-

3 CASEIN CONTENT OF HUMAN MILK 115 nodiffusion as described by Mancini (8) using mono-specific antibodies (Dakopatts, Copenhagen). Pure serum albumin, a-lactalbumin and secretory IgA (1 ls) were used as standards. In preliminary studies on whey samples, the concentration of 7S IgA in milk was determined to be too low (< 10% of total IgA) to interfere with the assay of secretory IgA. It has been shown earlier that secretory IgA constitutes more than 90% of total IgA in human milk (9). These proteins were assayed in skim milk samples, in isoelectric precipitates and in sediments. The values obtained for precipitates and sediments were subsequently subtracted from the pellet values to obtain a more true casein value. The casein content. Casein contents of milk samples were calculated in three ways: 1. Based on isoelectric precipitation: (Nitrogen in precipitate-16% of coprecipitated proteins) X Based on sedimentation experiments: (Nitrogen in sediment-16% of coprecipitated proteins) X Indirectly: Protein nitrogen X 6.25-(lactoferrin + human serum albumin + ct-lactalbumin + secretory IgA) Atomic absorption spectrophotometry. Calcium, magnesium, iron, copper and zinc were analyzed following a wet-ashing procedure using 16 N nitric acid (10). All glassware was acid-washed as described above. Flame atomic absorption spectrophotometry (IL 55 1, Norwalk, CT) was used and the instrument settings were those recommended by the manufacturer. For calcium and magnesium determinations 1% La(NO3)2 was added to reduce background interference. Amino acid analysis. Analysis was performed on a Durrum analyzer (Sunnyvale, CA, USA) after hydrolysis with 6 N hydrochloric acid at 1 10#{176}C for 24 h ( 1 1 ). Cysteine and methionine were analyzed after performic acid oxidation (12). Elecirophoresis. In order to evaluate the specificity of the sedimentation method, polyacrylamide gel electrophoresis was performed on ten samples of breast milk using a continuous system in urea. The concentration of acrylamide was 8% and that of bisacrylamide was 2% of the total acrylamide concentration (T8C2). Running buffer was 6 M urea in 0.9 M acetic acid at ph 2.5. Samples (1 50 each of casein and whey protein) were applied and gels were run at 2 ma/gel for 5 h at 4#{176}C.Following staining with Amido Black B, gels were destained with 0.9 M acetic acid. Statistical analyses. Analyses were carried out by several standard methods (13): linear regression was used to detect correlations; when significant differences were not found, analysis of variance was applied to linear regression. The different methods of estimating casein were compared pair-wise by a t- test for paired observations. Results The levels of the four major proteins (alactalbumin, lactoferrin, human serum albumin and secretory immunoglobulin A) as found in milk, isoelectric precipitates and sediments are shown in Table 1. The sum of the means of these proteins in the milk samples was 6.25 mg/mi or 67.4% of protein nitrogen X Of the four proteins, only lactoferrin was detected in the isoelectricprecipitate and the amount found corresponded to 12.9% of the lactoferrin present in the original milk samples. The corresponding analysis of the sediments showed traces of a-lactalbumin and levels of lactoferrin corresponding to 5.6% of the contents in the original milk. Gel electrophoresis of sediment and supernatant prepared by ultracentrifugation is shown in Figure 1. As can be seen, no casein subunits were observed in the supernatant (whey). The only whey protein that could be detected in the casein preparation was a- lactalbumin, and the amount was very small compared to that of casein. Total nitrogen, non-protein nitrogen, protein nitrogen and protein nitrogen X 6.25 in the milk samples are shown in Table 2. Mean total nitrogen was 2.07 mg/ml of milk and average non-protein nitrogen 0.60 mg/ml of milk corresponding to 29% of total nitrogen. The protein nitrogen was found to be 1.48 mg/ml of milk corresponding to an average crude protein content of 9.26 mg/ml. The

4 116 LNNERDAL AND FORSUM TABLE 1 Concentration of a-lactalbumin, lactoferrin, human serum albumin and secretory IgA in human milk presented in mg/ml milk and in % of protein N X Also shown are the levels of the same proteins collected in the isoelectric precipitates and sediments expressed in mg/ml milk and in % of the original protein concentration in milk Skim milk lsoelecthc precipitate Sediment % of milk protein % Of conc % Of conc Protein conc (N x 6.25) Protein conc in milk Protein conc in milk (mg/mi milk) (%) (mg/ml milk) (%) (mg/mi milk) (%) a-lactalbumin 2.56 ± 0.66* 27.7 ± 7.5 <.01 <0.01 <3 Lactoferrin 2.76 ± ± ± ± ± ± 1.7 Serum albumin 0.29 ± ± 0.5 <.01 - <.01 - Secretory IgA 0.63 ± ± 2.6 <.01 - <.01 - Sum of the means 6.25 ± ± 9.3 S Mean ± SD. nitrogen collected in the isoelectric precipitate and in the sediment corresponded to 28.4 and 20.9% of protein nitrogen, respectively; when the amount of nitrogen equivalent to the lactoferrmn found in the pellets were subtracted the corresponding percentages were 24.3 and 19.6, respectively. Casein values for nine individual milk II U. 0 IN FIG I. Polyacrylamide gel electrophoresis of human milk proteins in 6 M urea. Left: Sediment (casein); Right: Supernatant (whey proteins). samples as estimated by the three methods are shown in Table 3. Attempts were made to correlate data obtained by the different methods. The correlation coefficients obtamed were lower than 0.6 and were not significant. The sedimentation method showed the lowest mean value, 1.80 mg/ml while the highest mean was obtained by the indirect method, 2.96 mg/ml. The mean casein content obtained by isoelectric precipitation was 2.33 with a standard deviation of 1.69 which is high in comparison to the other two methods. Table 4 shows the results of the amino acid analysis performed on three of the milk samples. Apparently 17.5% of the amino acids (protein-bound and free) present in the original milk was sedimented by ultracentrifugation corresponding to a value of 27.6% found in the isoelectric precipitate. The standard deviation of individual and total amino acids recovered by the sedimentation method was small compared to the corresponding values obtained after isoelectric precipitation. The concentration and distribution of calcium, magnesium, zinc, copper and iron in whole milk, skim milk, supernatants, precipitates and sediments are shown in Table 5. A very small fraction of total milk calcium (6-10%) and magnesium (5%) was found in the isoelectric precipitates and sediments, respectively. Twenty-eight and 27% of the zinc and iron present in the milk was recovered in the sediment; corresponding figures for the isoelectric precipitate were 8 and 33%,

5 CASEIN CONTENT OF HUMAN MILK 117 TABLE 2 Partition of nitrogen in human milk expressed as mg/mi milk, % of total nitrogen and % of protein nitrogen Nitrogen mg/mi % Of total nitrogen % Of protein nitrogen Total nitrogen Non-protein nitrogen Protein nitrogen Nitrogen in isoelectric precipitate Nitrogen in isoelectric precipitate corrected for lactoferrin nitrogen Nitrogen in sediment Nitrogen in sediment corrected for lactoferrin nitrogen 2.07 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 7.0 C Mean ± SD. respectively. For copper similar amounts were recovered by the two methods, 1 7 and 21% of the total milk content, respectively. Discussion The average true protein content of the milk samples in this study, 9.26 mg/ml is similar to our previous estimate (14), 8.9 mg/ml by amino acid analysis. In the same study NPN was 0.41 mg/ml corresponding to 24% oftotal nitrogen which is in reasonable agreement with the present results although the concentrations of total nitrogen as well as non-protein nitrogen are higher in this group of samples as compared to our earlier data. The levels of whey proteins determined in this study are also in agreement with earlier results (15). It is apparent from the results of this study that quantitation of casein in breast milk is a complicated task. The three approaches gave different mean values for the casein content of breast milk and no correlation could be detected between them. The apparent conclusion is that the three methods measure, at least partly, different proteins. The reference method for casein determination, isoelectric precipitation, apparently includes in its estimates varying amounts of other proteins. This has been reported earlier by Nagasawa (16) and ourselves (5). Our experimental procedure in this study included a washing step, nevertheless other proteins were still found in the precipitate. The results of the isoelectric precipitation method also showed a considerable variation in comparison with the two other methods. Although it is possible that this reflects a true variation in the casein content of the samples it is also possible that this is due to varying amounts of coprecipitating whey proteins beyond those estimated in this study. For example, a-lactalbumin, serum albumin and immunoglobulins in human milk have isoelectric points near 4.6 (17) and are thus likely to precipitate with casein. Moreover, several whey proteins tend to aggregate, eg lactoferrmn and IgG (18, 19) and this tendency is increased at low ph. It is possible that this aggregation may reduce the antigenic determinants exposed to the antibodies, thereby potentially giving erroneously too low values for some co-precipitating whey proteins. However, it seems unlikely that this kind of phenomenon could explain more than a minor part of the differences found among the TABLE 3 Casein in individual milk samples estimated by isoelectric precipitation, sedimentation and an indirect method of analysis Sample A B C D E F G H I Mean SD By isoelectric precipitation I.69 Casein (mg/mi) determined By sedimentation Indirectly

6 118 LONNERDAL AND FORSUM TABLE 4 Amino acid concentration of three milk samples and in the corresponding sediments and isoelectric precipitates Sediment Isoelectric precipitate Milk (nmol/ml 5 Amount of (nmol/ml % Amount of (nmol/ml of milk) of milk) original milk of milk) original milk Aspartic acid 6524 ± ± ± ± ± 14.2 Threonine 3768 ± ± ± ± ± 16.0 Serine 4438 ± ± ± ± ± 15.4 Glutamic acid ± ± ± ± ± 18.0 Proline 7908 ± ± ± ± ± 28.8 Glycine 2938 ± ± ± ± ± 9.7 Alanine 4150 ± ± ± ± ± 12.8 Cysteine 1902 ± ± ± ± ± 4.4 Valine 4474 ± ± ± ± ± 25.1 Methionine 1078 ± ± ± ± ± 14.4 lsoleucine 3819 ± ± ± ± ± 20.3 Leucine 7214 ± ± ± ± ± 21.2 Tyrosine 2304 ± ± ± ± ± 21.6 Phenylalanine 1928 ± ± ± ± ± 17.0 Histidine 1486 ± ± ± ± ± 21.3 Lysine 4455 ± ± ± ± ± 15.8 Arginine 1871 ± ± ± ± ± 17.6 Total amino acids S Mean ± SD. methods. The observed variation could also The sedimentation method has been used be explained by an incomplete and irrepro- for preparative purposes rather than as an ducible precipitation of casein at ph 4.6. We analytical approach. According to our results have no data to support or deny this possi- less whey proteins were recovered in the bility. Thus, there are several questions re- pellet by this approach than with isoelectric garding the validity of the isoelectric precip- precipitation. This supports our hypothesis, itation method. indicated above, that coprecipitation of whey TABLE 5 Calcium, magnesium, zinc, copper and iron concentrations in whole milk, skim milk, sediments, precipitates and supernatants Skim milk Sedimentation experiment Itoelectric precipitation ( 5 of whole Sediment Supernatant Precipitate Supernatant Whole milk milk) (% of total) (% of total) (% of total) (% of total) Ca(mg/L) 292±8 269± ± ± ± ±10 (92%)t ( 10%)1: (90%4 (6%4 (94%4 Mg (mg/l) 29.8 ± 1. I ± ± ± ± ± 1.1 (101%)t (5%):$: (95%):1: (5%4 (95%)t Zn (mg/l) 1.16 ± ± ± ± ± ± 0.16 (85%)t (28%4 (72%)t (8%) (92%4 Cu (mg/l) 0.36 ± ± ± ± ± ± 0.07 (78%)t (l7%) (83%4 (2l%)t (79%)t Fe (mg/l) 0.32 ± ± ± ± ± ± 0.05 (84%)t (27%4 (73%)1: (33%):$: (67%4 S Mean ± SD. t % of total content in whole milk. t % of total content in supernatant and sediment/precipitate.

7 CASEIN CONTENT OF HUMAN MILK I 19 proteins is increased at low ph. The standard deviation observed for this method was low. It is possible that this method does not quantitatively estimate the casein present in human milk; however, gel electrophoresis of casein prepared by ultracentrifugation showed no significant quantities of whey proteins present in this sediment. The amount of casein applied was I 50 g; densitometric scannings indicated the amount of a-lactalbumin in the casein was close to the detection limit of -5 ;g, ie less than 3% of the total protein in the casein fraction. In addition, no casein subunits were detected in the resulting whey fraction (supernatant). Thus, the degree of overlap between the two protein fractions can be considered insignificant. Further experiments are needed to assess the sedimentation approach for its usefulness as a method for the estimation of casein. The sedimentation and isoelectnc precipitation methods indicate that the casein nitrogen of human milk constitutes % of total nitrogen rather than 40% which is regarded as the best current estimate. Our figures are supported by the indirect estimation of casein nitrogen which shows that 76.7% of total nitrogen could be accounted for by non-protein nitrogen and whey proteins leaving about 23% (.-3 mg protein/ml milk) that could possibly be casein nitrogen. As other proteins are known to be present in human milk, eg lysozyme, IgG, 1gM, and different enzymes, the true content of casein is probably even lower. It is difficult to estimate how much nitrogen to attribute to these minor proteins but from published levels of such components (20) they could be calculated to correspond to at least 2-4% of total nitrogen. This study thus suggests that the nitrogen compounds of breast milk are distributed in the following way: casein nitrogen:whey protein nitrogen:non-protein nitrogen = 20:50:30. This distribution would result in a ratio between casein nitrogen and whey nitrogen of 20:80. The values obtained for calcium, magnesium, zinc, copper and iron in human milk are similar to values reported earlier ( 19, 21). The lower contents of zinc, copper and iron in skim milk as compared to whole milk confirm our earlier findings that these elements are bound to the fat fraction of the milk. It should be noted that very little calcium is recovered in the casein fraction of human milk; this is in contrast to results obtained for cow s milk (22). The isoelectric precipitate and sedimentation methods used for isolation of casein resulted in preparations with different contents of zinc. This may be due to a relatively positive net charge of the casein precipitated at its isoelectric point which decreases its affinity for cations. Thus it may be important to carefully describe the method utilized for the preparation of casein when levels are given for the distribution of minerals between casein and whey in human milk. ci References 1. Rowland Si. The protein distribution in normal and abnormal milk. J Dairy Res 1938;9: Von Knoop E, Wortmann A. Zur Grossenverteilung der Caseinteilchen in Kuhmilch Ziegenmilch und Frauenmilch. Milchwissenschaft 1 960; 15: Macy 1G. Composition of human colostrum and milk. Am J Dis Child l949;78: L#{246}nnerdalB, Cederblad A, Sandstr#{246}mB. Low bioavailability of zinc from soy formula as compared to human milk and cow s milk formula. Am J Clin Nutr l983;37: L#{228}nnerdalB, Forsum E. Casein content of human milk. In: Hambraeus L ed, Nutrition in Europe. Proceedings of the third European nutrition conference. Stockholm, Sweden: Almquist & Wiksell International, 1980: Hambraeus L, Forsum E, Abrahamsson L, L#{246}nnerdal B. Automatic total nitrogen analysis in nitritional evaluations using a block digestor. Anal Biochem I 976;72: Laurell C-B. Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal Biochem l966;l5: Mancini G, Carbonara AO, Heremans JF. Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 1965;2: Goldman AS, Goldblum RM, Garza C, Nichols BL, O Brien Smith E. Immunologic components in human milk during weaning. Acta Paediatr Scand 1983:72: Clegg MS. Keen CL, L#{246}nnerdal B, Hurley LS. Influence of ashing techniques on the analysis of trace elements in animal tissue. I. Wet ashing. Biol Trace Elem Res 198 l;3:107-l Von Hofsten B, van Kley H, Eaker D. An extracellular proteolytic enzyme from a strain of arthrobacterium. II Purification and chemical properties of the enzyme. Biochim Biophys Acta 1965;l 10:

8 120 LONNERDAL AND FORSUM 12. Moore S. On the determination of cystine as cysteic acid. J Biol Chem 1963;238: Armitage P. Statistical methods in medical research. New York: Halsted Press, John Wiley & Sons mc, 1973, pp , and L#{246}nnerdalB, Forsum E, Hambraeus L. The protein content of human milk. I. A transversal study of Swedish normal material. Nutr Rep mt l976;l3:l L#{246}nnerdalB, Forsum E, Hambraeus L. A longitudinal study of the protein, nitrogen and lactose oontents of human milk from Swedish well-nourished mothers. Am J Clin Nutr l976;29: Nagasawa T, Kiyosawa J, Takase M. Lactoferrin and serum albumin of human casein in colostrum and milk. J Dairy Sci l974;57:l Jenness R. Protein composition of milk. In: Mc- Kenzie HA, ed. Milk Proteins: Chemistry and molecular biology. New York, Academic Press 1970;l Groves ML. Preparation of some iron-binding proteins and a-lactalbumin from bovine milk. Biochim Biophys Acta l965;loo: Fransson G-B, L#{246}nnerdalB. Iron in human milk. J Pediatr l980;96: Jenness R. The composition of human milk. Scm Perinatol l979;3: Fransson 0-B, Lonnerdal B. Copper, zinc, calcium and magnesium in human milk. J Pediatr 1982; 101: Fransson 0-B, L#{246}nnerdal B. Distribution of trace elements and minerals in human milk and cow s milk. Pediatr Res 1983;17:912-5.