CHAPTER 7

Mineralsf Ions, and Trace

Elements in Milk
A. Ioni c Interaction s i n Mil k
MARGARET C. NEVILLE
PEIFANG ZHANG
JONATHAN C . ALLEN

I. Introductio n
Knowledge of the structural and electrochemical compartmentalizatio n o f
the ioni c component s o f mil k i s importan t t o th e understandin g o f io n
secretion, ion absorptio n fro m th e gastrointestina l tract , and ionic effect s
on th e propertie s o f foo d product s derive d fro m milk . Certai n ions ,
sodium, potassium , an d chlorid e exis t largel y i n th e ionize d stat e i n th e
aqueous compartmen t o f milk . Th e other , divalen t cation s o f mil k ar e
distributed amon g th e structura l compartment s an d protei n component s
in a highly specific manner. Most, including calcium, magnesium, and zinc,
have measurabl e concentration s o f fre e io n an d ar e par t of th e comple x
electrochemical equilibriu m depicte d i n Figure 1 for calcium and magnesium. Many of these ions also are bound with very high affinity t o specific
milk proteins. I n this chapter we focus on both the ionic equilibria in the
aqueous compartmen t o f mil k an d th e distributio n o f ion s amon g it s
structural compartments. We will begin by considering the methodologie s
available for measurement of these parameters. It will then be necessary to
consider mil k p H an d th e majo r hydroge n io n buffer s tha t regulat e it .
Monovalent ions will be briefly considered; then the compartmentalization
and equilibria of the divalent cations and the anions to which they bind will
be discussed at some length. I n general, our discussion will be confined t o
H A N D B O O K O F MIL K COMPOSITIO N 5 7
Copyright O I99 S b y Academi c Press , Inc.
All right s reserved . N o reproductio n withou t permission .

578 Margare

t C. Neville et al.
CaHPO^ Casei

n Micelle

.2+ Ca^ *

Citrate'- /

/'caHCO,

^ O , ^<5:^H

CaCitrate H

X*^^*^^
'
COj ^

Figure I Principl e interaction s o f calciu m an d magnesiu m i n th e aqueou s compartmen t

of milk .

human an d bovin e miik , th e tw o species fo r whic h substantia l dat a ar e

available. Calcium and zinc will receive a good bi t of attention becaus e of
their nutritiona l importanc e an d the complexity o f their interactions .

il. Methodologie s
A. Analysis of Ionic Distribution in the Structural
Compartments of Milk
The structura l compartments of milk have been describe d in Chapter 2 as
consisting of the milk fat, the cellular compartment, th e aqueous compart ment, casein, and the membrane o r "fluff compartment . Thes e compart ments ar e best separate d b y centrifugation a s illustrated i n Figur e 2 . A
20-min centrifugation a t 6000g" separates the cream an d cellular fraction s
from fres h whol e milk. The aqueous infranatant i s carefully remove d using
a needle and syringe to avoid disturbing the loose cream layer . An aliquot
is reserved fo r analysis and the remainder subjecte d t o ultracentrifugatio n
at 100,000g " for 1 or 2 hr (4'*C ) to bring dow n th e casein micelle s whic h
form a solid pellet in the bottom of the tube. A loose pellet, called the fluff,
directly abov e th e casei n contain s th e so-calle d ski m mil k membrane s
(Huston an d Patton , 1986) . A thi n lipi d laye r o n th e surfac e contain s
residual milk fat. If necessary, the infranatant ca n be centrifuged throug h
a 10,00 0 M W cutoff filte r t o separate ioni c specie s boun d t o macromolecules. If this step is used it is important t o determine tha t the ionic species
in questio n doe s not bind t o the filter.
Although i t i s possibl e t o analyz e th e crea m an d casei n fraction s
directly fo r thei r ioni c content , becaus e thes e fraction s ar e difficul t t o
handle i t is much simple r t o analyze onl y th e aqueous an d fluff fraction s

579

7. Minerals , Ions , an d Trac e Element s i n Mil k

Centrifugation

Ultrafiltration

Figure 2 Protoco l fo r separatio n o f structura l compartment s o f huma n milk . Th e first

centrifugation i s carried ou t a t roo m temperature , th e secon d a t 4C . To preven t proteas e
action an d bacteria l growth , 0.0 1 ^l g of sodiu m azid e an d 5 0 ^i l of a solutio n o f proteas e
inhibitors (15 mg/ml of iodoacetamide an d 1 5 mg/ml of phenylmethy l sulfony l fluoride) ar e
added t o each mil k sample. After measuremen t o f pH , small aliquot s are take n fo r analysi s
of tota l lipi d b y th e creamatocrit (Luca s et al., 1978 ) or othe r convenien t method . Anothe r
aliquot i s put asid e fo r th e io n concentratio n i n whol e milk , and th e remainde r i s subjected
to th e centrifugatio n protoco l shown . T o determin e io n bindin g t o macromolecula r com ponents o f th e aqueou s phas e afte r ultracentrifugatio n (I2) , th e fluid ca n b e subjecte d t o
centrifugation throug h a 10,00 0 M W cutoff filter. Th e filtrate contain s fre e ion s an d ion s
bound t o smal l molecula r weigh t component s o f milk .

using th e followin g equation s t o calculat e th e ioni c concentration s i n th e

cream an d casei n fractions . Th e equation s als o correct fo r th e presenc e o f
aqueous infranatan t i n th e fluff fraction . Th e assumption s mad e i n thes e
calculations ar e (i ) tha t th e infranatan t (/j ) compositio n afte r th e first
centrifugation reflect s los s o f th e fa t fraction , (ii ) tha t th e cellula r fractio n
is to o smal l t o affec t ioni c distribution , an d (iii ) tha t afte r th e high-spee d
centrifugation th e composition of the infranatant (/2 ) reflects th e loss of th e
casein an d fluff fractions . Th e amoun t o f solut e i s referre d t o it s concen tration i n whole milk . Solut e associate d wit h th e fluff fractio n i s calculate d
directly fro m analysi s o f thi s fractio n correctin g fo r th e presenc e o f
aqueous supernatan t (/g) . Th e unit s o f th e quantitie s resultin g fro m thes e
calculations ar e mmo l o r gram s o f io n pe r lite r o f whol e milk .
Cation associate d wit h th e fa t fractio n i s
[Sf] = [ W ] - [ / , ] ( ! - / O ,

[1]

where [S{] is th e catio n associate d wit h th e fat , [W] i s the concentratio n i n

whole milk , [/, ] i s th e catio n concentratio n i n th e infranatan t afte r th e
6000g centrifugation, an d F is the fractio n o f fa t in the whole mil k sample .
Cation associate d wit h th e casei n an d fluff fractions , Sp , i s
[5p] = ( [ / , ] - [ / 2 ] ) ( l - F ) .

[2]

580

Margaret C . Nevill e e t al.

Cation associate d wit h the aqueous fraction , S^q,

IS

[Saq] = [W ] - [Sf] - [5p] .

[3]

Cation associated wit h the fluff fraction , S^ , i s

[ s ] = ([Fi ] - [/2])(y) ,

[4]

where V is the volume of the fluff fraction divided by the initial volume of
the mil k sample.
Cation associated wit h casein is
[5]

[5c] = [Sp ] - [S^] .

B. Analysis of Electrochemical Equilibria
In the Aqueous Compartment

In order to determine the electrochemical interactions of any ionic species

in milk i t is necessary t o kno w the ioni c activity, th e concentrations o f all
potential ligands , an d th e equilibriu m constant s fo r th e relevan t interac tions. Table I gives the total concentration o f the relevant ionic species in
human and bovine milk and Table I I gives the equilibrium constants and
equations for the interactions of these species. Zinc is not included in Table
I because its concentration in milk is highly dependent on the duration of

TABLE I
Major ioni c Constituents of Human and Bovine Miik^
Units

Human mil k

Bovine mil k

Sodium

mmol/liter

6.3

24.2

Component

Potassium

mmol/liter

13.9

34.7

Chloride

mmol/liter

11.6

30.2

Magnesium, tota l

mmol/liter

1.8

5.1

mmol/liter

7.5

29.4
2.0*

Calcium, total
Calcium, ionize d

mmol/liter

3.0

Citrate

mmol/liter

2.6

9.2

Phosphate, fre e

mmol/liter

1.8

11.2

Bicarbonate

mmol/liter

6.0

4.8

g/liter

18.0

261

6.8

6.7

Casein
pH

''Values for composition of human milk at 90 days postpartum from Allen et a/. (1991) except
casein which i s taken fro m Case y and Hambidg e (1983) . Values fo r bul k her d bovin e milk
from Whit e and Davie s (1958).
*Value obtained b y calculation usin g a series of assume d equilibriu m constant s (Hol t et al.,
1981). The murexid e metho d gav e values closer to 3.0 (Hol t et al., 1981).

581

7. Minerals , Ions , and Trace Element s i n Mil k

TABLE I I
Equations for Ionic Equilibria in Milk
Equilibrium constan t

Products/reactants

ky,

-13.357

[H + ] X [OH-]/[HOH ]

-10.585

([H-^] X [HCOs-lVpCO s

ks
k.

-10.059

([H-^] X [CO3-MHCO3- ]

-3.029

([Citrate-] x [H-^])/[citrate ]

k.

-4.580

([Citrate^-] x [H-^])/[citrate- ]

ke

-6.102

([Citrate^-] x [ H + ])/[citrate2-]

kj

-2.131

([H2PO4-] X [H-])/[H3P04 ]

ks

-7.255

([HPO42-] X [H + MH2PO4-]

fh

-12.104

([PO43-] X [H^])l[H?0^-]
[Ca.citrate-]/(Ca2+] x [citrate^-] )

4.606

2.536

[Ca.citrate]/(Ca2 + ] x [citrate^-] )

1.250

[Ca.citrate-^]/(Ca2-^] x [citrate"] )

0.840

[Ca.H2P04'^]/([Ca2-^] x [H2PO4- ]

2.401

[Ca.HP04]/([Ca2-^] x [HPO42- ]

LI

4.72

[Zn.citrate-]/(Zn2 + ] x [citrate^-] )

L2

3.72

[Zn.citrate]/(Zn2-^] x [citrate^-] )

L6

4.446

[Mg.citrate-]/(Mg2 + ] x [citrate2-] )

L7

2.21

[Mg.citrate]/(Mg2-^] x [citrate2-] )

L8

2.529

[Mg.HP04]/([Mg2-] X [HPO42-]

Note; Equilibrium constants are expressed as logjo of the constant for the reaction in the forward direction. All constants are corrected to the ionic strength of human milk (\k = 0.0 3 M).
Subscripted letters represent the arbitrarily chosen designation for the equilibrium constant.

lactation (Case y et al., 1989) . The concentratio n o f ionized specie s can be

determined usin g eithe r ion-selectiv e electrode s o r equilibrium dialysis .
Highly specifi c ion-selectiv e electrode s ar e available fo r hydroge n ion,
sodium, potassium, and calcium. It has been necessary to use equilibrium
dialysis t o determine th e concentration o f free zin c i n milk fo r reasons
discussed belo w (Zhan g and Allen , 1992) .
Older methods for measuring Ca^"^ and Mg^" ^ activity included resi n
equilibrium (Christianso n et ai, 1954 ) and dye binding wit h murexid e
(Tessier an d Rose, 1957) . Thes e method s wer e compare d wit h a n ion selective electrode method and gave higher values for [Ca^^] in milk (Holt
et ai, 1981) . Th e low readings fro m th e electrode wer e though t b y th e
authors to be a matrix effect; some electrode systems may be more sensitive
to matri x effect s tha n others . Also , alteration s o f th e calcium comple x
formation b y either the resin or the murexide i s a possibility.
If resins or dyes are to be used for measuring the activity of metals, the
resin or dye should have an appropriate binding and adequate sensitivity.

582 Margare

t C . Nevill e et al.

Allen and Zhang (unpublished) attempted to use these methods to measure

zinc activity ([Zn^-^]). However, the cation-exchange resins tested had such
high zinc affinity that they disrupted the equilibrium in the milk and bound
most of the zinc. Lower-affinity resin s did not have a high enough affinit y
and selectivity t o bind a measurable quantit y o f zin c in the presenc e o f a
1000-fold excess of calcium. Similar problems occurred with metal-sensitive
dyes. A n equilibriu m dialysi s approac h usin g th e colloida l calciu m phos phate of bovine casein as the zinc complexing agent proved successful. The
zinc content of this material, placed inside a dialysis bag, is proportional to
the zinc activity of the dialysate (McGann et ai, 1983) . Zinc chelators must
be used in the dialysate to achieve reliable and reproducible zin c activities
in the nanomolar range and to diminish th e effect o f zin c contamination.
Milk sample s shoul d b e take n an d maintaine d unde r anaerobi c con ditions for measurement of pH and the activity of certain ions for reasons
described under Section III . We have described an appropriate techniqu e
for huma n mil k (Alle n an d Neville , 1983) .

III. Hydroge n Io n Equilibria in Milk

The followin g reactio n i s at equilibrium i n milk:
CO2 + H2O ^ //2CO 3 :; //^ -f HCO3 - ;; 2//-^ + CO 3 =. [6
In all mammals th e body fluids, including milk , ar e equilibrated wit h 5%
CO2. When milk is removed from the breast or udder and exposed t o the
air, CO 2 i s lost , th e equatio n shift s t o th e left , an d th e p H rises . Th e
decreased hydroge n io n als o shift s th e reaction , H ^ - f HC03-^2H ^ +
C03 = , to the right, increasing [CO =3]. Because of the high [Ca^^] in milk
the increase d [00^3 ] probabl y als o result s i n formatio n o f a CaCO s
complex, explainin g decrease d [Ca ^ + J a s mil k i s store d an d pC0 2
decreases.
The bicarbonate concentration i n human milk can be calculated fro m
the p H an d pC0 2 t o vary between abou t 4.3 an d 6.0 mmol/lite r (Alle n et
aL, 1991). Equilibrated with a pC02 of 40 mm Hg, the pH of human milk
as present in the breast is 6.6 to 6.8. Measured values are often given as 7.2
or above because anaerobic precaution s are not usually take n and considerable loss of CO2 occurs particularly when milk is expressed with a breast
pump. We have been unable to find values for the pH of bovine milk taken
under condition s i n whic h th e bicarbonate-C0 2 equilibriu m wa s main tained unde r in vivo conditions.

IV. Distributio n of Monovalent Ion s in Milk

Measurement o f th e tota l concentratio n o f sodiu m an d potassiu m i n
human milk by ion-selective electrodes or by flame photometry gave equal

7. Minerals , Ions , an d Trac e Element s In Mil k 58

values (Neville et al, 1984) , implying that these ions are present as the free
ionic species . A simila r conclusio n wa s reache d b y Hol t et al. (1981 ) fo r
bovine milk . Th e assumptio n i s usuall y mad e tha t chlorid e i s als o com pletely ionized. At the ionic strength of milk, there may be a small amount
of interactio n o f chlorid e wit h sodium , calcium , magnesium , an d potas sium, amountin g t o les s tha n 5 % of th e tota l i n bovin e mil k (Hol t et al,
1981). A similar conclusion applies to human milk with its even lower ionic
strength. Th e concentratio n o f thes e ion s i n mil k ultrafiltrate s (Hol t an d
Jenness, 1984 ) is also consistent wit h the conclusion tha t monovalent ion s
are present i n free solutio n i n th e aqueous compartment o f mil k only.

V. Distributio n of Divalent Cation s among

the Structural Compartment s o f Mil k
The centrifugation protoco l illustrate d i n Figure 2 was used t o determine
the divalent cation distribution amon g th e compartments o f huma n milk.
The results are shown in Table III. Calcium was found only in the aqueous
compartment and associated with casein. Contrary to earlier reports from
the literatur e (Nevill e et al, 1985 ; Lonnerda l an d Fransson , 1981) , n o
calcium was associated with the fat fraction. This is expected because milk
fat globules originate fro m a cellular compartment wit h a submicromolar
calcium concentration. When human milk samples were frozen and thawed
prior t o analysis , 6. 0 2.1% o f th e calciu m wa s associated wit h th e lipid ,
suggesting that damage t o the milk fat globule membran e during expression or storage ma y increase calciu m bindin g eithe r t o free fatt y acid s or
to mil k fa t globul e membran e fraction s (Riieg g an d Blanc , 1982) . Blak e
and Hennin g (1988 ) observe d tha t negligible calciu m wa s associated wit h
washed cream in rat milk suggesting tha t the same principl e ma y hold in
other species.

TABLE II I
structural CompartmentaKzatio n o f Divalen t Ion s In Human Milk ^

Milk fraction

Calcium
(mmol/hter)

Magnesium
(mmol/liter)

Copper
(jimol/liter)

Zinc
(jimol/liter)

Whole milk
Aqueous
Fat
High-speed pelle t
Membranes

7.96 0.46
6.76 0.35
-0.02 0.23
1.150.20
0.08 0.02

1.59 0.08
1.38 0.09
0.11 0.09
-0.07 0.04
0.17 0.05

3.34 0.50
1.51 0.22
0.890.19
1.180.24
0.16 0.04

26.78 4.37
12.05 2.66
8.131.94
7.26 1.98
0.65 0.09

''All concentrations expresse d pe r liter of whol e milk.

584

Margaret C. Neville et al .

Although magnesiu m wa s foun d mainl y i n th e aqueou s compartmen t

of human mil k (Figur e 3 ) significant amount s were also associated wit h th e
lipid and membranou s compartments ; non e wa s associated wit h the casei n
pellet. Coppe r an d zin c wer e mor e o r les s evenl y distribute d betwee n th e
aqueous, lipid , an d casei n fractions . Anothe r grou p foun d tha t casei n
micelles i n huma n mil k containe d 14 % of th e tota l zinc , seru m albumi n
bound 28% , 29 % wa s foun d t o b e presen t i n th e aqueou s compartment ,
and th e remainin g 29 % was associate d wit h th e fat , possibl y boun d t o th e
alkaline phosphatas e i n th e mil k fa t globul e membran e (Lonnerda l et al,,
1982; Hurle y an d Lonnerdal , 1982 ; Lonnerda l an d Fransson , 1981) .
Extrinsic labelin g studie s (Sandstro m et al, 1983 ) demonstrate d tha t
zinc bioavailabilit y wa s 28 % fro m huma n milk , 25 % fro m whey-adjuste d
cow's mil k formula , 15 % fro m cow' s milk , an d 10 % fro m soy-base d
formula. I t is reasonable t o conclude tha t it is not only th e amoun t o f zinc ,
but als o th e compound s bindin g th e elemen t tha t affec t th e degre e t o
which i t i s absorbed . I n contras t t o huma n milk , practicall y al l th e zin c i n
bovine mil k wa s i n th e ski m mil k fractio n (Blakesboroug h et al, 1983) .
Casein micelle s i n bovin e mil k separate d b y ultracentrifugatio n (100,000g for 1 hr) containe d abou t 90 % o f th e tota l zinc ; onl y 10 % was associate d
with th e solubl e phas e (Parkas h an d Jenness, 1966 ; Blakesboroug h et al,
1983; Singh etal, 1989b) . About half of the soluble zinc was nondialyzable ,
indicating tha t i t wa s tightl y boun d t o protei n (Sing h et al, 1989b) . Thus ,
only abou t 5 % o f tota l zin c i n cow' s mil k shoul d b e associate d wit h
small-molecular-weight ligands .
Soluble macromolecules , includin g nonmicella r casein , remai n i n th e
infranatant afte r high-spee d centrifugation . I n orde r t o determin e
whether divalent ions are bound t o these milk components, th e infranatan t
can b e force d throug h a 10,00 0 M W cutof f filter. Result s o f suc h a n
100
Calcium

ed
-

o
H
O
O

0-.

Aqueous Fa t Casei n Fluf f

Structural Compartmen t
Figure 3 Distributio n of divalent cations among the structural compartments of human milk.
Milk sample s wer e fractionate d accordin g t o th e protoco l i n Figur e 2 and th e whol e milk ,
infranatant, an d membran e fractio n analyze d fo r calcium , magnesium , copper , an d zin c by
atomic adsorption spectroscop y as described previousl y (Casey et al, 1989 ; Allen et al, 1991) .

7. Minerals , Ions , and Trace Element s i n Mil k

585

experiment for the calcium of human mil k suggested tha t approximately

13% of the calcium in the infranatant, or 1 mmol/liter, was retained in the
macromolecular fraction (Neville etal, 1994) . Added to the 15% of calcium
associated wit h casein , a total o f 28 % of th e calcium i n huma n mil k is
associated with macromolecules, and 72 % is free or associated with smallmolecular-weight components. I n a very careful stud y Arnaud and Favier
(1992) foun d tha t 2 0 12% o f th e zin c i n huma n wa s present i n the
ultrafiltrable fractio n at all stages of lactation. These values are consistent
with the earlier wor k of Lonnerdal et al (1982).

VI. Calciu m and Zinc Binding to Casein

Figure 4 show s a comparison o f the distribution o f calcium amon g the
structural compartment s o f human an d bovine milk . Abou t 65 % of the
calcium is associated wit h casei n in bovine milk . Th e difference betwee n
bovine and huma n mil k is due to a number of factors: (i) in bovine milk
the concentration of casein is nine times that of human milk (Table I); (ii )
in bovin e mil k som e o f the citrate i s associated wit h th e casein micell e
(Farrell, 1988 ) so that some of the protein-associated calcium may be in the
form of calcium citrate. In human milk only about 0.1 mmol/liter of citrate
is not ultrafiltrable (Hol t and Jenness, 1984) . (iii) Finally, huma n casein is
not fully phosphorylated (Groves and Gordon, 1970) . The calcium binding
capacity o f human casei n ca n be calculated t o be only abou t 1 4 mol o f
calcium/mol of casein (Nevill e et a/., 1994) ; in most species the ratio is 2 0
mol of calcium/mol o f casein (Jenness , 1979) .
The casei n micelle s i n bovine mil k are composed o f subunits linke d
together by colloidal calciu m phosphat e an d hydrophobic bondin g (Slat tery, 1976 ; Schmidt, 1982) . Parkash and Jenness (1966 ) reported tha t the
zinc i n bovine casei n micelle s i s presen t i n two forms, on e of whic h is
loosely bound and is readily removed by dialysis against dilute EDTA (< 2

?3

0i

S2 0
o
U

O Ultra filtrate

i S Pa t

10

V//////////y
^7777777^.

Human Bovin

^ Protein ,
other
H i Casei n

Figure 4 Compariso n of structural compartmentation o f calcium in human and bovine milk.

Bovine mil k value s take n fro m th e data o f Griffin ei al (1988) . Figur e fro m Nevill e ei al.
(1994). Use d by permission o f iht Journal of Dairy Science.

586 Margare

t C . Nevill e e t al.

mm) or extractio n wit h dithiazone i n acetone. Singh an d co-worker s (Sing h

et al, 1989b ) foun d tha t 32 % o f th e zin c i n bovin e ski m mil k wa s directl y
bound t o caseins , whil e abou t 63 % wa s associate d wit h colloida l calciu m
phosphate.
Zinc binding by whole bovine and huma n casei n and b y purified bovin e
casein an d whe y protein s wa s investigated b y equilibriu m dialysi s b y Sing h
and co-worker s (Sing h et ai, 1989a) . Ther e wer e larg e difference s i n th e
estimated number s o f bindin g site s o n th e differen t caseins : 11 , 8, an d 2
atoms zinc/mo l fo r bovin e a s p , P- , an d x-casein , respectively . T h e zinc binding capacities of the individua l bovine caseins, i.e., a^r > P - > x-casein ,
were i n th e sam e orde r a s thei r phosphoserin e content s whic h ar e 8 i n
Qsi-casein, 5 i n p-casein , an d 1 i n x-casei n (Ribadea u et al, 1972 ; Gro sclaude et a/., 1973) . Dephosphorylatio n o f bovin e whol e casei n markedl y
reduced it s zinc-bindin g capacity . Thes e result s sugges t tha t th e phospho serine group s o f th e casei n ar e th e primar y bindin g site s fo r zin c (Harze r
and Kauer , 1982 ; Sing h et al, 1989a) . However , i t appear s tha t casei n
contains zinc-bindin g site s othe r tha n phosphoserin e residue s becaus e th e
total molecule s o f zin c boun d (-^11 ) exceede d th e numbe r o f phospho serine residue s (~8 ) an d dephosphorylatio n o f casei n di d no t eliminat e it s
zinc-binding capacity . Experiment s i n ou r laborator y wit h dialysi s an d
chelators suggeste d tha t th e zin c i n colloida l calciu m phosphat e i s i n
equilibrium wit h th e fre e zin c i n th e dialysat e o r milk .
Casein fro m huma n mil k ha d a slightly highe r zinc-bindin g capacit y ( 7
or 8 atom s Zn/mo l protein ) tha n whol e bovin e casei n (50 6 atom s Zn/mo l
protein), but th e apparen t associatio n constant s wer e th e sam e (Sing h et al,
1989b), indicatin g a similarit y i n th e natur e o f zin c bindin g t o th e phos phoserine residue s i n casei n fro m thes e tw o species . T h e abov e researc h
shows that , o n a n equimola r basis , the zinc-bindin g capacit y an d affinit y o f
whole huma n casei n ar e simila r t o thos e o f whol e bovin e casein . T h e larg e
difference betwee n th e casei n concentration s o f bovin e an d huma n mil k
probably account s fo r th e highe r proportio n o f zin c associated wit h casei n
micelles o f cow* s milk . Huma n mil k contain s littl e o r n o colloida l calciu m
phosphate (Jenness , 1973) . Wit h th e exceptio n o f bovin e seru m albu min, whic h boun d ove r 8 atom s Zn/mol , th e bovin e whe y proteins , P lactoglobulin, a-lactalbumin , an d lactoferri n ha d littl e capacit y fo r zin c
binding (Sing h et al, 1989b) .

V I I . Divalen t Catio n Equilibri a i n th e

Aqueous Compartmen t o f Mil k
A. Calcium
Calcium i s pumpe d int o th e saccule s o f th e termina l Golg i apparatu s an d
secretory vesicle s of th e mammar y alveola r cel l by a n ATPas e (Nevill e an d

7. Minerals , Ions , and Trac e Element s i n Mil k 58

Walters, 1983) . Ther e i t interact s wit h casein , citrate , phosphate , bicar bonate, an d carbonat e leadin g t o th e formatio n o f casei n micelles . Al though equilibriu m i s attained , th e calciu m i n al l th e fraction s appear s t o
be readily exchangeable because all fractions of milk equilibrated wit h ^^Ca
within 4 h r (Nevill e an d Keller , unpublished ; se e als o Sandstro m et al.,
1983). The equation s given in Table I I can be solved numericall y using any
one of a number of moder n computer programs . When th e concentration s
of th e variou s ion s presen t i n huma n mil k a t 3 month s lactatio n an d th e
measured ionize d calciu m ( 3 mM ) ar e use d a s independen t variables , th e
only significan t calciu m salt s ar e foun d t o b e calciu m citrat e ( 1 mM ) an d
calcium phosphat e (0. 4 mM ; Tabl e IV) .
It i s instructiv e t o examin e th e compositio n o f huma n mil k durin g
lactogenesis. I n human s ther e i s a delay o f abou t 2 day s after birt h befor e
the onse t o f copiou s mil k secretio n take s place ; a majo r volum e increas e
takes plac e o n Day s 3 an d 4 postpartu m (Nevill e et aL, 1988 ; se e als o
Chapter 3A) . Figur e 5 depict s th e concentration s o f th e relevan t compo nents of mil k over this period. Total calciu m nearl y double d betwee n Day s
1 an d 3 postpartu m (se e als o Ken t et ai, 1992) , althoug h ionize d calciu m
actually fel l slightly . Magnesiu m an d p H remaine d relativel y constant ;
changes i n thes e parameter s canno t accoun t fo r th e observe d change s i n
total calcium . Citrat e an d phosphat e ros e i n paralle l wit h th e calciu m
suggesting tha t the increase i n these tw o anions was largely responsibl e fo r
the increas e i n tota l calciu m i n earl y lactation . T o determin e whethe r thi s
was indeed th e case, we calculated th e amounts of calcium bound t o citrate
TABLE I V
Calculated Values for Majo r ioni c Forms of Calcium and Magnesium
in Human an d Bovine Milk

Ionic species Huma

Calcium
[Ca2 + ] 3.
[CaCit-] 2.
[CaP04] 0.
Magnesium
[Mg2+] 0.9
[MgCit-] 0.8
[MgP04] 0.0

n milk (mmol/liter)' Bovin

e mil k (mmol/liter)*

0 2.
0 6.
4 0.

0
9
6

4 0.

2 2.
3 0.

0
3

"Values for huma n mil k fro m Nevill e et al. (1994) an d unpublishe d dat a fro m Nevill e and
Allen and calculated using data from human milk at 3 months postpartum. Ionize d calcium
was measured on anaerobic samples using an ionized calcium electrode as described. All other
values were calculated fro m th e equations i n Table II .
^Values for bovine milk taken from Hol t et al. {19S\). These ma y be subject to reevaluation
because ionize d calcium was derived b y calculation an d loss of CO 2 from th e mil k was not
considered.

S88

Margaret C. Neville et al.

Days Postpartu m
Figure 5 Change s in the concentration of total calcium and other milk components involved
in calcium equilibri a durin g lactogenesis . Dat a replotte d fro m thos e o f Nevill e et al. (1991).
Figure fro m Nevill e et al. (1994). Used b y permission o f the Journal of Dairy Science.

and phosphat e fo r eac h day . Th e result s ar e show n i n Figur e 6 . O n Da y

1 of lactation nearly all the calcium can be accounted fo r as ionized calcium .
By Day s 3 an d 4 abou t 6 mmol/lite r o f th e calciu m i s presen t a s ionize d
calcium, calcium phosphate , an d calciu m citrate , an d abou t 2 mmol/lite r i s
presumed t o b e boun d t o casein . Unfortunately , ther e ar e n o accurat e
measurements o f th e casei n concentratio n i n huma n mil k durin g lactoge nesis. However , Patto n et al. (1986 ) showe d clearl y tha t casei n i s ver y lo w
prior t o da y 2 afte r whic h i t increase s rapidly . W e assume , therefore , tha t
the shade d are a largel y represent s calciu m boun d t o casein .
In late lactation th e concentration o f calciu m i n human mil k (Figur e 7 ,
left) decline s significantly fro m nearl y 7 mmol/liter a t 15 0 days postpartu m

Measured Tota l
Calcium
a
o^

It
j3 w

P J^ Calciu

I,

m Phosphat e

Days Postpartu m
Figure 6 Calciu m equilibria during lactogenesis. The lines are calculated using the measured
milk compositio n show n i n Figur e 5 an d th e equation s i n Tabl e II . Th e shade d are a
represents calciu m no t accounte d fo r b y the equilibriu m calculations , presumabl y boun d t o
protein. Figur e from Nevill e et al. (1994). Used b y permission o f ihe Journal of Dairy Science.

589

7. Minerals , Ions , an d Trac e Element s I n Mil k

PH
Total Calcium

56

1 =< *"N Citrate

^2

a
Magnesium

Phosphate

200 30

0 40 0 50 0

Days Postpartum

Total Calcium

"6

i2

f CaPhospbate

a^
200 30

0 40

0 50 0

Days Postpartum

Figure 7 Huma n mil k component s an d calciu m equilibri a i n lat e lactatio n (al l value s i n
mmol/liter). Mil k composition extrapolate d fro m Nevill e eM/. (1991) ; calculations carrie d ou t
as those fo r Figur e 6 . All subjects wer e producin g mor e tha n 40 0 m l of milk/day . Figur e fro m
Neville ei al. (1994) . Use d b y permissio n o f tht Journal of Dairy Science.

to about 4.5 mmol/liter at 450 days. The question of which components of

milk ar e responsibl e fo r th e declin e i s answere d b y solvin g th e electro chemical equation s o f Tabl e II . The result s o f thi s analysis are show n in
Figure 7 (right). Clearly, the fall in calcium is not due to a change in ionized
calcium, which actually increases slightly over this time. Rather, both citrate
and th e protei n fractio n represente d b y the shaded are a decrease. Thes e
results, together with the observations during lactogenesis, suggest that the
total concentration of calcium in human milk is not a regulated variable in
the physiologica l sense , bu t varie s a s a function o f th e concentration s o f
citrate and caseins in the milk. The finding that the calcium in cow's milk
also varie s wit h th e citrat e concentration (Hol t an d Muir , 1979 ) suggest s
that this migh t be a general phenomenon .
B. Magnesium

As show n i n Tabl e II I abou t 18 % of th e magnesiu m i n huma n mil k i s

associated wit h the lipid and membran e fractions . Th e remainder , i n the
aqueous fraction , i s foun d t o b e divide d amon g fre e magnesium , mag nesium citrate , an d magnesiu m phosphat e (Tabl e IV ) whe n th e equa tions of Table I I are solved. Hol t et al. (1981 ) obtained a similar result for
bovine milk .
C. Zinc

Using severa l zin c chelator s t o prepar e standar d solution s o f know n

[Zn2+], the [Zn2+ ] of bovine skim milk was found b y equilibrium dialysis
to be approximately 5 x 10"^ ^ Af (Zhang and Allen, 1992) . Studies of the

590 Margare

t C . Nevill e et al.

binding of zinc in milk ultrafiltrates have been very controversial. Although

both critic and picolinic acids have been proposed as low-molecular-weight
ligands, the concentration of the complexes depends on the concentrations
of the ligands, the free zinc, and the concentrations of competing ligands.
Using the value for [Zn^-^^] obtained by Zhang and Allen (1992), the citrate
concentration in Table I , and the association constants from Table II , the
concentration of the zinc-citrate complex in bovine milk is only about 0.68
mM, or about 0.02% of th e total .
Picolinic acid has been implicated i n the zinc absorption process . Zinc
picolinate was found t o be efficacious whe n fed t o children with disorders
which responde d t o zin c therap y (Krieger , 1980) . However , th e picolini c
acid concentration i n human milk is very low, less than 3.7 \iM (Rebello et
al, 1982 ) a s measure d wit h high-performanc e liqui d chromatography .
This low concentration together with the low zinc activity would appear to
rule out any important role for this compound as a zinc complexing ligand
in human milk . Computer simulations use d to determine th e distribution
of zin c amon g low-molecular-weigh t ligands , namel y citrate , glutamate ,
and picolinate, i n both huma n an d bovine mil k (Ma y et al, 1982 ) showed
that at high concentration s o f picolinate , thi s ligand ma y for m a neutra l
complex, whic h coul d facilitat e intestina l absorptio n o f th e metal , bu t at
lower levels of picolinate, such as those found in milk, the concentration of
zinc picolinate woul d b e vanishingly small .
VIII. Summar y an d Conclusion s
In considering the ionic interactions in milk it is important to know how the
ion i n questio n i s compartmentalize d i n th e structura l compartment s a s
well a s th e concentration s o f al l interactin g specie s i n th e aqueou s com partment. Th e activit y o f th e fre e specie s shoul d b e measure d usin g a n
ion-selective electrode, if available, or equilibrium dialysis under conditions
in which the CO2 content of the milk can be controlled. When this analysis
is carried out for the calcium content of human milk it becomes clear that
the ionized calcium in milk is more or less constant at about 3.0 mmol/liter.
Physiologic variations in the total calcium are due primarily to changes in
citrate an d casei n durin g th e cours e o f lactation . Recen t result s usin g
equilibrium dialysis to determine the free zinc suggest that the majority of
zinc, i n bovin e mil k a t least , i s boun d t o high-molecular-weigh t species .
Similar experiments remai n t o be carried ou t wit h huma n milk .
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