The American Journal ofCiinical Nutrition 41: JANUARY 1985, pp 1 13-120. Printed in USA© 1985 American Society for Clinical Nutrition

Original Research Communications-methods

Casein content of human 1,2

Bo L#{246}nnerdal and Elisabet Forsum

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 wheyproteins and subtracting these from the total protein content). Gel electrophoresis and amino acidanalysis were used to confirm some of the results. The casein content (mg/ml) of mature humanmilk (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 caseinN: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 whileisoelectric 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 findingsshould be considered with regard to infant nutrition. Am J Clin Nuir 1985;l 13-120.

KEY WORDS Human milk, casein, whey, whey proteins, iron, copper, zinc, calcium,magnesium, trace elements, minerals

Introduction

The traditional method for classifying milkproteins was developed by Rowland (1) andintended primarily for bovine milk proteins.According to this approach, casein is precip-itated at its isoelectric point, pH 4.6. Proteinsremaining in solution are called whey pro-

teins. This procedure has also been appliedto human milk, but it was recognized earlythat human casein differed from cows’ caseinin its physico-chemical properties, ie, theprecipitate formed by human casein at pH4.6 was looser and softer than that formedby bovine casein.

Casein as a class of proteins consists ofseveral subunits (a, � and sc-casein) whichform micelles with Ca� and P04 givingmilk its characteristic white appearance.There are differences between the caseinsfrom different species, however, eg the mi-celles in human milk are considerably smallerthan those of bovine milk (2). Also, the

presence of a-casein in human milk has not

been clearly demonstrated. No method hasbeen developed to satisfactorily quantitatethe content of casein in human milk.

Using isoelectric precipitation human ca-sein nitrogen has been estimated to compriseabout 35% of total nitrogen in human milk(3). A figure of4O% has been widely accepted

and is used as a guideline by infant formulamanufacturers when cow’s milk protein con-tent is modified for infant feeding. This mod-

ification involves adjustment of the ratio ofcasein nitrogen to whey protein nitrogen incow’s milk (80:20) to mimic the ratio found

I From the Department of Nutrition, University of

California, Davis, CA 95616 and the Department ofMedical Nutrition, Huddinge University Hospital, Ka-rolinska Institute, 5-141 86 Huddinge, Sweden.

2 Address reprint requests to: Bo L#{246}nnerdal, Depart-ment of Nutrition, University of California, Davis, CA95616.

Received April 13, 1984.Accepted for publication July 24, 1984.

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114 LONNERDAL AND FORSUM

in human milk (40:60). In this way, a morebalanced amino acid profile of the formulais obtained. Such modifications may also

affect the bioavailability of trace minerals.For example, Lonnerdal et al (4) presenteddata indicating the bioavailability of zinc islower from a formula with a casein N:wheyN ratio of 80:20 than from a formula with acorresponding ratio of 40:60 suggesting that

zinc in cow’s milk may be less availablewhen bound to casein than when present inthe whey fraction of milk. Modifications in

the ratio of casein to whey proteins of cow’smilk should thus be evaluated for the nutri-tional consequences to the formula-fed infant.Since the casein:whey ratio may affect traceelement bioavailability it becomes interestingto know the mineral distribution in humanmilk as a guideline when the optimal ratiobetween casein and whey in formulas isconsidered.

In an earlier study (5) it was found thatover a pH-range of 4.2-5.4 similar amountsof protein-nitrogen precipitated from humanmilk; significant amounts of this precipitatewere non-casein proteins, eg, lactoferrin. Also,by an indirect approach (ie by estimating thetotal amount of nitrogen in breast milk, non-protein nitrogen and the contribution of ni-trogen from individual whey proteins) caseinnitrogen was calculated to be considerablylower than 40% of breast milk nitrogen. Inthis study the partition of nitrogen betweentotal nitrogen, non-protein nitrogen, proteinnitrogen and individual whey proteins hasbeen investigated in nine human milk sam-pIes and the amount of nitrogen equivalentto the theoretically maximal content of caseinnitrogen has been calculated. Also studiedwere the amounts of nitrogen precipitated atpH 4.6 and of nitrogen-containing materialsedimented by ultracentrifugation. Further-more, the amounts of calcium, magnesium,zinc, copper and iron recovered in casein arereported as prepared by the ultracentrifuga-tion and isoelectric precipitation methods.

Materials and methods

Milk samples. Samples were obtainedfresh from healthy, exclusively breast-feedingSwedish donors after 4-16 weeks of lactation.

The protocol was approved by the HumanSubjects Committee at University of Uppsala.The milk was collected at the mid-morningmeal (8-10 AM) and was expressed by amanual breast pump (Gentle Flow, Eklundand Co. Davis, CA). All parts of the pumpin contact with the milk and the plasticstorage bottles had been thoroughly washed,soaked in 10% nitric acid and rinsed withdistilled deionized water. All samples weredefatted by centrifugation at 4,000 g for 30 mmat 4#{176}C.

Isoelectric precipitation. Milk samples (20ml) were adjusted to pH 4.6 by slow additionof hydrochloric acid (1 M) during stirring at20#{176}C.After 1 h, the samples were centrifugedat 10,000 g for 15 mm at 20#{176}C.Supernatantswere decanted and the pellets suspended insaline (5 ml) and adjusted to pH 4.6 withhydrochloric acid. Following 1 5 mm of stir-

ring, the suspended pellets were centrifugedas above. The resulting supernatants werecombined with the previous supernatants andthe pellets (isoelectric precipitates) were dis-solved in saline (5 ml) adjusted to pH 8.0with sodium hydroxide.

Sedimentation. Milk samples (1 5 ml) wereultracentrifuged (MSE superspeed 75 Mea-suring & Scientific Equipment Ltd., London)at 105,000 g for 30 mm at 20#{176}C.Supernatants

were decanted and the sediments were dis-solved in saline (5 ml) adjusted to pH 8.0

with sodium hydroxide.Protein nitrogen (PN) and non-protein ni-

trogen (NPN). Estimations were as follows: 2ml 24% trichloroacetic acid was added to 2ml milk and the samples were centrifuged at27,000 g for 60 mm after which the super-natants were decanted and their nitrogencontent analyzed (NPN). The precipitateswere dissolved in 0. 1 M NaOH and analyzedfor nitrogen (PN).

Nitrogen analysis. Analysis was performedby a modified micro-Kjeldahl method de-scribed earlier (6). Milk samples and dissolvedprecipitates and sediments were analyzed fortotal nitrogen. For conversion of nitrogen toprotein the factor of 6.25 was used.

Individual proteins. Lactoferrin was deter-mined by immunoelectrophoresis accordingto Laurell (7). Serum albumin, ct-lactalbuminand IgA were determined by radial immu-

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CASEIN CONTENT OF HUMAN MILK 115

nodiffusion as described by Mancini (8) usingmono-specific antibodies (Dakopatts, Copen-

hagen). Pure serum albumin, a-lactalbuminand secretory IgA (1 lS) were used as stan-

dards. In preliminary studies on whey sam-ples, the concentration of 7S IgA in milk wasdetermined to be too low (< 10% of total

IgA) to interfere with the assay of secretoryIgA. It has been shown earlier that secretory

IgA constitutes more than 90% of total IgAin human milk (9). These proteins were

assayed in skim milk samples, in isoelectricprecipitates and in sediments. The values

obtained for precipitates and sediments weresubsequently subtracted from the pellet valuesto obtain a more “true” casein value.

The casein content. Casein contents ofmilk samples were calculated in three ways:

1 . Based on isoelectric precipitation:(Nitrogen in precipitate-16% of copre-

cipitated proteins) X 6.25

2. Based on sedimentation experiments:(Nitrogen in sediment-16% of coprecip-

itated proteins) X 6.253. Indirectly:

Protein nitrogen X 6.25-(lactoferrin + hu-

man serum albumin + ct-lactalbumin

+ secretory IgA)

Atomic absorption spectrophotometry. Cal-

cium, magnesium, iron, copper and zincwere analyzed following a wet-ashing proce-

dure using 16 N nitric acid (10). All glasswarewas acid-washed as described above. Flame

atomic absorption spectrophotometry (IL55 1, Norwalk, CT) was used and the instru-ment settings were those recommended bythe manufacturer. For calcium and magne-sium determinations 1% La(NO3)2 was addedto reduce background interference.

Amino acid analysis. Analysis was per-

formed on a Durrum analyzer (Sunnyvale,CA, USA) after hydrolysis with 6 N hydro-chloric acid at 1 10#{176}Cfor 24 h ( 1 1 ). Cysteineand methionine were analyzed after performic

acid oxidation (12).Elecirophoresis. In order to evaluate the

specificity of the sedimentation method,polyacrylamide gel electrophoresis was per-

formed on ten samples of breast milk usinga continuous system in urea. The concentra-tion of acrylamide was 8% and that of bis-

acrylamide was 2% of the total acrylamideconcentration (T8C2). Running buffer was

6 M urea in 0.9 M acetic acid at pH 2.5.Samples (1 50 � each of casein and wheyprotein) were applied and gels were run at 2mA/gel for 5 h at 4#{176}C.Following stainingwith Amido Black B, gels were destainedwith 0.9 M acetic acid.

Statistical analyses. Analyses were carriedout 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 estimat-ing casein were compared pair-wise by a t-

test for paired observations.

Results

The levels of the four major proteins (a-

lactalbumin, lactoferrin, human serum al-

bumin and secretory immunoglobulin A) asfound in milk, isoelectric precipitates andsediments are shown in Table 1 . The sum ofthe means of these proteins in the milksamples was 6.25 mg/mi or 67.4% of proteinnitrogen X 6.25. Of the four proteins, onlylactoferrin was detected in the isoelectric-precipitate and the amount found corre-

sponded to 12.9% of the lactoferrin presentin the original milk samples. The corre-sponding analysis of the sediments showedtraces of a-lactalbumin and levels of lacto-ferrin corresponding to 5.6% of the contentsin the original milk.

Gel electrophoresis of sediment and super-natant prepared by ultracentrifugation is

shown in Figure 1 . As can be seen, no caseinsubunits were observed in the supernatant(whey). The only whey protein that could bedetected in the casein preparation was a-

lactalbumin, and the amount was very smallcompared to that of casein.

Total nitrogen, non-protein nitrogen, pro-tein 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 andaverage non-protein nitrogen 0.60 mg/ml ofmilk corresponding to 29% of total nitrogen.The protein nitrogen was found to be 1.48mg/ml of milk corresponding to an averagecrude protein content of 9.26 mg/ml. The

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116 L�NNERDAL AND FORSUM

TABLE 1Concentration of a-lactalbumin, lactoferrin, human serum albumin and secretory IgA in human milk presented inmg/ml milk and in % of protein N X 6.25. Also shown are the levels of the same proteins collected in theisoelectric precipitates and sediments expressed in mg/ml milk and in % of the original proteinconcentration in milk

Skim milk lsoelecthc precipitate Sediment

% ofmilk

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 <3Lactoferrin 2.76 ± 0.59 29.8 ± 2.2 0.35 ± 0.14 12.9 ± 5.9 0.15 ± 0.06 5.6 ± 1.7Serum albumin 0.29 ± 0.04 3.1 ± 0.5 <.01 - <.01 -

Secretory IgA 0.63 ± 0.36 6.8 ± 2.6 <.01 - <.01 -

Sum of themeans 6.25 ± 1.39 67.4 ± 9.3

S Mean ± SD.

nitrogen collected in the isoelectric precipitateand in the sediment corresponded to 28.4and 20.9% of protein nitrogen, respectively;when the amount of nitrogen equivalent to

the lactoferrmn found in the pellets were sub-tracted the corresponding percentages were24.3 and 19.6, respectively.

Casein values for nine individual milk

IIU.

0

INFIG 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 methodsare shown in Table 3. Attempts were madeto correlate data obtained by the differentmethods. The correlation coefficients ob-tamed were lower than 0.6 and were notsignificant. The sedimentation methodshowed the lowest mean value, 1 .80 mg/mlwhile the highest mean was obtained by theindirect method, 2.96 mg/ml. The meancasein content obtained by isoelectric precip-itation was 2.33 with a standard deviation of

1.69 which is high in comparison to theother two methods.

Table 4 shows the results of the aminoacid analysis performed on three of the milksamples. Apparently 17.5% of the aminoacids (protein-bound and free) present in theoriginal milk was sedimented by ultracentri-

fugation corresponding to a value of 27.6%found in the isoelectric precipitate. The stan-dard deviation of individual and total amino

acids recovered by the sedimentation methodwas small compared to the correspondingvalues obtained after isoelectric precipitation.

The concentration and distribution of cal-cium, magnesium, zinc, copper and iron inwhole milk, skim milk, supernatants, precip-

itates and sediments are shown in Table 5.

A very small fraction of total milk calcium(6-10%) and magnesium (5%) was found inthe isoelectric precipitates and sediments,respectively. Twenty-eight and 27% of thezinc and iron present in the milk was recov-ered in the sediment; corresponding figuresfor the isoelectric precipitate were 8 and 33%,

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CASEIN CONTENT OF HUMAN MILK 117

TABLE 2Partition 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 nitrogenProtein nitrogen

Nitrogen in isoelectric precipitateNitrogen in isoelectric precipitate

corrected for lactoferrinnitrogen

Nitrogen in sedimentNitrogen in sediment corrected

for lactoferrin nitrogen

2.07 ± 0.30

0.60 ± 0.041.48 ± 0.29

0.42 ± 0.25

0.36 ± 0.270.31 ± 0.08

0.29 ± 0.08

29.0 ± 4.071.0 ± 4.0

20.3 ± 13.1

18.0 ± 13.615.0 ± 4.7

14.0 ± 4.4

28.4 ± 19.8

24.3 ± 20.120.9 ± 7.1

19.6 ± 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 issimilar to our previous estimate (14), 8.9mg/ml by amino acid analysis. In the same

study NPN was 0.41 mg/ml correspondingto 24% oftotal nitrogen which is in reasonableagreement with the present results althoughthe concentrations of total nitrogen as wellas non-protein nitrogen are higher in thisgroup of samples as compared to our earlier

data. The levels of whey proteins determinedin 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 isa complicated task. The three approaches

gave different mean values for the caseincontent of breast milk and no correlationcould be detected between them. The appar-ent conclusion is that the three methodsmeasure, at least partly, different proteins.

The reference method for casein determi-nation, isoelectric precipitation, apparentlyincludes in its estimates varying amounts of

other proteins. This has been reported earlierby Nagasawa (16) and ourselves (5). Ourexperimental procedure in this study included

a washing step, nevertheless other proteinswere still found in the precipitate.

The results of the isoelectric precipitationmethod also showed a considerable variationin comparison with the two other methods.

Although it is possible that this reflects a truevariation in the casein content of the samplesit is also possible that this is due to varyingamounts of coprecipitating whey proteinsbeyond those estimated in this study. Forexample, a-lactalbumin, serum albumin andimmunoglobulins in human milk have iso-electric points near 4.6 (17) and are thuslikely to precipitate with casein. Moreover,several whey proteins tend to aggregate, eglactoferrmn and IgG (18, 19) and this tendencyis increased at low pH. It is possible that thisaggregation may reduce the antigenic deter-minants exposed to the antibodies, therebypotentially giving erroneously too low valuesfor some co-precipitating whey proteins.However, it seems unlikely that this kind ofphenomenon could explain more than a mi-nor part of the differences found among the

TABLE 3Casein in individual milk samples estimated byisoelectric precipitation, sedimentation and anindirect method of analysis

Sample

Casein (mg/mI) determined

Byisoelectric

precipitation

By sedi-

mentationIn-

directly

ABCDEFGHI

4.671.051.502.160.931.53

-

1.894.91

2.201.851.361.182.492.341.851.691.23

3.132.564.083.502. 1 13.231.402.024.66

MeanSD

2.33I .69

1.800.48

2.961.08

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118 LONNERDAL AND FORSUM

TABLE 4Amino 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 ± 789 865 ± 165 13.2 ± 1.0 1299 ± 840 21.0 ± 14.2Threonine 3768 ± 361 597 ± 89 15.8 ± 0.8 864 ± 536 23.8 ± 16.0Serine 4438 ± 431 670 ± 98 15.1 ± 1.0 1013 ± 659 24.1 ± 15.4Glutamic

acid 1281 1 ± 474 2251 ± 337 16.8 ± 0.9 3465 ± 2288 27.2 ± 18.0Proline 7908 ± 706 2093 ± 237 26.4 ± 0.6 3238 ± 2163 42.3 ± 28.8Glycine 2938 ± 446 280 ± 68 9.5 ± 1.0 401 ± 248 14.6 ± 9.7Alanine 4150 ± 628 592 ± 120 14.2 ± 0.8 896 ± 444 22.8 ± 12.8Cysteine 1902 ± 262 104 ± 37 5.4 ± 1.2 123 ± 74 6.8 ± 4.4Valine 4474 ± 545 990 ± 204 22.0 ± 2.0 1557 ± 1028 36.6 ± 25.1Methionine 1078 ± 169 134 ± 23 12.5 ± 0.5 204 ± 133 20.4 ± 14.4lsoleucine 3819 ± 416 729 ± 106 19.0 ± 0.8 1094 ± 721 29.9 ± 20.3Leucine 7214 ± 841 1399 ± 204 19.4 ± 0.6 2154 ± 1413 31.3 ± 21.2Tyrosine 2304 ± 191 466 ± 87 20.1 ± 2.1 680 ± 465 30.7 ± 21.6Phenylalanine 1928 ± 286 289 ± 42 15.1 ± 1.2 444 ± 289 24.5 ± 17.0Histidine 1486 ± 142 289 ± 40 19.4 ± 0.9 454 ± 298 31.7 ± 21.3Lysine 4455 ± 447 625 ± 100 14.6 ± 0.8 1005 ± 660 23.5 ± 15.8Arginine 1871 ± 374 321 ± 92 17.0 ± 1.4 430 ± 277 25.2 ± 17.6

Total aminoacids 8148 799 1428 227 17.5 1.1 2167 1415 27.6 18.6

S Mean ± SD.

methods. The observed variation could also The sedimentation method has been usedbe explained by an incomplete and irrepro- for preparative purposes rather than as anducible precipitation of casein at pH 4.6. We analytical approach. According to our resultshave no data to support or deny this possi- less whey proteins were recovered in thebility. Thus, there are several questions re- pellet by this approach than with isoelectricgarding the validity of the isoelectric precip- precipitation. This supports our hypothesis,itation method. indicated above, that coprecipitation of whey

TABLE 5Calcium, magnesium, zinc, copper and iron concentrations in whole milk, skim milk, sediments,precipitates and supernatants

Sedimentation experiment Itoelectric precipitation

Skim milk

(‘5 of whole Sediment Supernatant Precipitate SupernatantWhole milk milk) (% of total) (% of total) (% of total) (% of total)

Ca(mg/L) 292±8� 269± 10 25.8±4.1 233± 11 15.8±7.2 260±10(92%)t ( 10%)1: (90%4 (6%4 (94%4

Mg (mg/L) 29.8 ± 1 . I 30. 1 ± 1.8 1.4 ± 0.2 27. 1 ± 1.0 1.4 ± 0.5 29.0 ± 1.1(101%)t (5%):$: (95%):1: (5%4 (95%)t

Zn (mg/L) 1.16 ± 0.19 0.99 ± 0.17 0.30 ± 0.04 0.75 ± 0.12 0.08 ± 0.02 0.90 ± 0.16(85%)t (28%4 (72%)t (8%)� (92%4

Cu (mg/L) 0.36 ± 0.07 0.28 ± 0.08 0.06 ± 0.01 0.29 ± 0.06 0.06 ± 0.02 0.24 ± 0.07(78%)t (l7%)� (83%4 (2l%)t (79%)t

Fe (mg/L) 0.32 ± 0.05 0.27 ± 0.16 0.08 ± 0.03 0.21 ± 0.06 0.10 ± 0.08 0.20 ± 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.

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CASEIN CONTENT OF HUMAN MILK I 19

proteins is increased at low pH. The standarddeviation observed for this method was low.

It is possible that this method does notquantitatively estimate the casein present inhuman milk; however, gel electrophoresis ofcasein prepared by ultracentrifugation showedno significant quantities of whey proteinspresent in this sediment. The amount of

casein applied was I 50 �g; densitometricscannings indicated the amount of a-lactal-

bumin in the casein was close to the detectionlimit of ‘-5 ;�g, ie less than 3% of the totalprotein in the casein fraction. In addition,

no casein subunits were detected in the re-sulting whey fraction (supernatant). Thus,the degree of “overlap” between the twoprotein fractions can be considered insignif-icant. Further experiments are needed to

assess the sedimentation approach for itsusefulness as a method for the estimation ofcasein.

The sedimentation and isoelectnc precipi-

tation methods indicate that the casein nitro-gen of human milk constitutes 1 5-20% oftotal nitrogen rather than 40% which is re-garded as the best current estimate. Ourfigures are supported by the indirect estima-tion of casein nitrogen which shows that

76.7% of total nitrogen could be accountedfor by non-protein nitrogen and whey pro-teins leaving about 23% (‘�‘.-3 mg protein/ml

milk) that could possibly be casein nitrogen.As other proteins are known to be present inhuman milk, eg lysozyme, IgG, 1gM, and

different enzymes, the true content of casein

is probably even lower. It is difficult toestimate how much nitrogen to attribute tothese minor proteins but from publishedlevels of such components (20) they could

be calculated to correspond to at least 2-4%of total nitrogen. This study thus suggeststhat the nitrogen compounds of breast milk

are distributed in the following way: caseinnitrogen:whey protein nitrogen:non-proteinnitrogen = 20:50:30. This distribution wouldresult in a ratio between casein nitrogen andwhey nitrogen of 20:80.

The values obtained for calcium, magne-sium, zinc, copper and iron in human milkare similar to values reported earlier ( 19, 21).The lower contents of zinc, copper and ironin skim milk as compared to whole milk

confirm our earlier findings that these ele-ments are bound to the fat fraction of the

milk. It should be noted that very littlecalcium is recovered in the casein fraction ofhuman milk; this is in contrast to resultsobtained for cow’s milk (22). The isoelectricprecipitate and sedimentation methods usedfor isolation of casein resulted in preparations

with different contents of zinc. This may be

due to a relatively positive net charge of thecasein precipitated at its isoelectric pointwhich decreases its affinity for cations. Thus

it may be important to carefully describe the

method utilized for the preparation of caseinwhen levels are given for the distribution ofminerals between casein and whey in human

milk. ci

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