The Structure of Milk: Implications For Sampling and Storage

CHAPTER 2
The Structure of Milk:

Implications for Sampling
and Storage
A. Th e Mil k Lipi d Globul e Membran e
THOMAS W. KEENA N
STUART PATTO N
I. Intracellula r Origi n an d Growth o f Milk Lipi d

Globules
Membrane an d membrane-associate d materia l whic h surround s th e

triacylglycerol-rich mil k lipi d globule s commonl y i s referred t o as the mil k
fat o r mil k lipi d globul e membran e (MLG M hereafter) . Thi s materia l
originates fro m specialize d region s o f apica l plasm a membran e o f mam -
mary epithelia l cells , an d fro m endoplasmi c reticulu m (ER ) an d perhap s
other intracellula r compartments . Tha t portio n o f th e MLG M derive d
from apica l plasm a membrane , terme d th e primar y membrane , ha s a
typical bilaye r o r uni t membran e appearance , wit h a n electron-dens e
material o n th e inne r membran e face . Tha t componen t derive d fro m E R
lacks bilaye r membran e structure , primaril y i s compose d o f protein s an d
polar lipids , an d cover s th e surfac e o f th e lipi d droplet s withi n th e cell .
Constituents o f thi s coa t materia l mediat e intracellula r fusion s throug h
which droplet s gro w i n volum e an d als o ma y b e involve d i n interactio n o f
droplets wit h plasm a membrane .
H A N D B O O K O F MIL K COMPOSITIO N
Copyright © 199 5 b y Academi c Press , Inc.
All righ u reserved . N o reproductio n withou t permission .
6 Thoma s W. Keena n and Stuart Patto n
A. Droplet Formation
Earliest intracellula r precursor s o f mil k lipi d globule s appea r t o originat e

from ER . Triacylglycerols appea r to accumulate a t focal point s on or in th e
ER membran e (Dylewsk i et a/. , 1984) . Whethe r thi s accumulatio n o f tri -
acylglycerols i s du e t o localize d synthesi s o r accretio n i s unknown . I t ha s
been suggeste d tha t triacylglycerol s accumulat e betwee n th e halve s o f th e
bilayer membrane an d are released fro m E R into the cytoplasm as droplets
coated with th e oute r o r cytoplasmic hal f o f th e E R membrane (Lon g an d
Patton, 1978 ; Scow et ai, 1980) . Som e morphologica l evidenc e supportin g
this suggestio n ha s bee n obtaine d (Patto n an d Keenan , 1975 ; Zacze k
and Keenan , 1990) , bu t informatio n tha t woul d prov e o r disprov e thi s
hypothesis i s lacking .
B. Growt h of Drop/ets
By whateve r mechanis m the y originate , mil k lipi d globul e precursor s first

appear i n th e cytoplas m a s smal l (diameter s < 0. 5 \im) droplet s tha t hav e
a triacylglycerol-ric h cor e surrounde d b y a granula r coa t materia l lackin g
unit-like (o r bilaye r membran e structure , bu t tha t i n localize d region s
appears thickened , wit h tripartite-lik e structur e (Dylewsk i et al.^ 1984 ;
Deeney et aL, 1985) . Smal l lipi d droplets , terme d microlipi d droplets ,
appear t o gro w i n volum e b y fusion s wit h eac h other . Fusion s giv e ris e t o
larger droplets, termed cytoplasmi c lipi d droplets, operationally define d a s
those droplet s wit h diameter s > 1 fxm.
In addition t o observations mad e by electro n microscopi c examinatio n
of fixed an d sectione d material , th e natur e o f th e surfac e coa t materia l o n
intracellular lipi d droplet s ha s bee n explore d throug h isolatio n an d com -
positional analysi s o f droplet s (Dylewsk i et ai, 1984 ; Deene y et ai, 1985) .
Droplets ca n b e isolate d b y densit y gradien t centrifugation , takin g advan -
tage o f th e fac t tha t the y hav e lowe r densitie s tha n d o organelle s an d
vesicles derive d fro m component s o f th e endomembran e system . Droplet s
ranging fro m < 1 to 1.1 2 g/c c i n densit y hav e bee n characterized ; densit y
was inversely relate d t o volume . Droplet s o f differen t densit y classe s fro m
cow mammar y glan d ha d lipi d t o protei n ratio s rangin g fro m abou t 1.5: 1
to 40:1 .
Triacylglycerols wer e th e majo r lipi d clas s i n droplet s o f al l sizes .
Surface coa t materia l o f droplet s containe d cholestero l an d th e sam e five
major phospholipi d classe s foun d i n milk : sphingomyeli n an d th e phos -
phoglycerides of choline, ethanolamine, inositol , and serine. Lipid droplet s
also ha d monohexosyl - an d dihexosylceramide s an d ganglioside s i n thei r
surface coa t material ; thes e glycosphingolipid s ar e know n constituent s o f
milk lipi d globules .
When separate d i n sodiu m dodecylsulfate-polyacrylamid e gel s (SDS -
PAGE), micro - an d cytoplasmi c lipi d droplet s ha d comple x an d virtuall y
2. Th e Structur e o f Mil k 7
identical polypeptid e patterns . Man y polypeptide s wit h electrophoreti c

mobilities identica l t o thos e o f intracellula r lipi d droplet s ar e foun d i n
MLGM material . Severa l polypeptide s o f MLG M an d intracellula r lipi d
droplets shar e antigeni c reactivity .
In summary , morphologica l observation s an d biochemica l dat a ar e
consistent wit h a n E R origi n o f intracellula r lipi d drople t precursor s o f
milk lipid globules. The materia l on th e surface o f lipid droplets within th e
cell appear s t o remai n associate d wit h th e droplets , a t leas t i n part , whe n
they ar e secrete d a s mil k lipi d globules .
II. Rol e of Intracellular Lipi d Drople t Coa t

Material
Coat materia l o n surface s o f intracellula r lipi d droplet s undoubtedl y i s

required t o stabiliz e th e triacylglycerol-ric h cor e o f droplet s an d preven t
their coalescenc e i n th e cytoplasm . Beyon d thi s stabilizatio n role , th e coa t
material appear s t o participat e als o i n drople t fusions , an d i n droplet -
plasma membran e interactions . I f cytoskeleta l element s functio n i n guid -
ing lipi d droplet s fro m thei r site s o f origi n t o thei r site s o f secretio n fro m
the cell , coa t constituent s ma y participat e i n interactio n wit h element s o f
the cytoskeleton. Mechanism s responsibl e fo r unidirectiona l transi t of lipi d
droplets throug h th e cytoplas m t o apical cel l regions , fro m whic h the y ar e
secreted, ar e no t know n wit h certainty . Evidenc e tha t microtube s o r mi -
crofilaments ma y b e involve d i n thi s proces s ha s bee n obtained , bu t
evidence contradictin g thes e interpretation s als o ha s bee n obtaine d (dis -
cussed i n Mathe r an d Keenan , 1983) . A s yet , w e hav e n o clear , definitiv e
information o n wha t i s responsible fo r thi s unidirectiona l transfe r o f lipi d
droplets. I n th e mil k o f cows , lipi d globule s rang e i n siz e fro m unde r 0. 2
to ove r 1 0 fx m in diameter . Small globule s (belo w 1 fim) ar e mos t numer -
ous, accounting fo r 80 % or more of th e total number of globules, but thes e
small globule s accoun t fo r les s tha n 10 % of th e tota l volum e o f mil k fat .
Globules wit h diameter s betwee n 1 and 8 î m contain 90 % or mor e o f th e
total volume of mil k fat. Larg e droplets are few i n number, but account fo r
1 to 3% of th e fa t volume o f mil k (reviewe d i n Brunner, 1965 ; Mulder an d
Walstra, 1974) . Dat a availabl e sugges t a simila r siz e rang e o f globule s i n
milks o f human s (Riieg g an d Blanc , 1981) . Globul e siz e distributio n i s
discussed unde r Sectio n V .
Within th e cell , on e mechanis m fo r growt h o f lipi d droplet s appear s
to b e fusion s o f microlipi d droplet s with eac h othe r t o for m large r drop -
lets. Microlipi d droplet s ca n als o fus e wit h larger , cytoplasmi c lipi d drop -
lets, providin g triacylglycerol s fo r continue d growt h i n volum e o f large r
droplets (Dylewsk i et aL, 1984) ; thi s growt h i s pronounce d i n globule s
in apica l cel l region s (Stemberge r an d Patton , 1981 , 1984) . Whil e image s
interpreted a s microlipi d droplet-microlipi d drople t an d microlipi d
droplet-cytoplasmic lipi d drople t fusion s ar e commonl y see n i n electro n

micrographs, severa l investigator s hav e faile d t o find morphologica l evi -
dence fo r fusion s betwee n larger , cytoplasmi c lipi d droplet s (Wooding ,
1971a; Stemberge r an d Patton , 1981 , 1984 ; Dylewsk i et ai, 1984) .
From researc h t o dat e th e siz e rang e o f lipi d globule s i n mil k ca n b e
accounted fo r b y the fusio n process . Smalle r mil k lipi d globule s aris e mos t
probably fro m secretio n o f microlipi d droplet s tha t hav e undergon e
no o r onl y a fe w fusions . Large r droplet s originat e b y continue d fusion s
with microlipid droplets . Morphologica l an d kineti c evidence i n support o f
this interpretation ha s been obtained. However , thi s evidence i s insufficien t
to allo w th e interpretatio n tha t fusio n o f droplet s i s th e sol e o r majo r
mechanism fo r drople t growth . Othe r possibl e mechanism s fo r thi s
growth, fo r example , lipi d transfe r protein s whic h conve y triacylglycerol s
from thei r sit e o f synthesi s t o growin g lipi d droplets , canno t b e exclude d
(Patton, 1973) .
The proces s o f microlipi d drople t fusio n ha s bee n reconstitute d i n a
cell-free syste m (Valivulla h et ai, 1988) . Fusio n appear s t o involv e constit -
uents o f th e surfac e coa t o f lipi d droplets . A s droplet s grow , exces s coa t
material i s los t fro m th e surface , a s woul d b e expecte d fro m geometri c
consideration o f surfac e are a (are a = inr^) t o volum e (volum e = 4/3jir^ )
ratios o f wha t ar e essentiall y spherica l particles . Th e fat e o f coa t materia l
shed fro m drople t surface s durin g fusio n withi n cell s i s unknown . I n th e
cell-free system , fusio n wa s promote d b y calcium, b y a protein fractio n o f
cytosol, an d b y ganglioside s o f th e surfac e coa t o f lipi d droplets . I n
agreement wit h morphologica l observation s o f section s fro m cells , i n th e
cell-free syste m microlipi d droplet-microlipi d drople t an d microlipi d
droplet-cytoplasmic lipi d drople t fusion s occurred , bu t cytoplasmi c lipi d
droplet-cytoplasmic lipi d drople t fusion s di d not . Th e reason s wh y cyto -
plasmic lipi d droplet s d o no t fus e wit h eac h othe r i s no t apparent . Withi n
the scop e o f compositiona l analyse s performe d t o date , coa t material s o n
micro- an d cytoplasmi c lipi d droplet s largel y ar e indistinguishable , excep t
for th e increase d leve l o f ganglioside s pe r uni t protei n i n cytoplasmic lipi d
droplets.
III. Mil k Lipi d Globule Secretio n
The proces s o f lipi d drople t secretio n ha s bee n describe d repeatedl y sinc e

Bargmann an d Knoo p (1959 ) originall y observe d tha t droplet s becam e
surrounded b y apica l plasm a membran e a s the y wer e budde d fro m
cells (reviewe d i n Patto n an d Keenan , 1975 ; Mathe r an d Keenan , 1983 ;
Keenan et al, 1988) . Woodin g (1971a ) provide d morphologica l evidenc e
for a n alternativ e mechanism , on e i n whic h fa t droplet s contactin g
the apica l plasm a membran e als o becom e surrounde d with secretor y
vesicles tha t fus e wit h eac h othe r an d th e plasm a membrane . Thi s
resulted i n formatio n o f intracellula r vacuole s containin g membrane -

coated lipi d droplets . Releas e o f droplets , surrounde d partiall y i n apica l
plasma membrane , wa s envisione d t o occu r b y emptyin g o f th e vacuola r
contents. Morphologica l evidenc e fo r whic h o f thes e alternativ e processe s
occurs or predominate s i s equivocal. Mos t biochemical evidenc e favor s th e
interpretation tha t th e majo r mechanis m fo r secretio n o f mil k lipi d glob -
ules involve s envelopmen t o f droplet s directl y i n plasm a membrane . A
minor contributio n fro m Golg i apparatus-derive d secretor y vesicl e mem -
brane cannot be excluded (reviewe d i n Mathe r and Keenan , 1983 ; Keena n
etai, 1988) .
Plasma membran e region s wit h whic h lipi d droplet s associat e ar e
characterized b y the appearance o f an electron-dense materia l on the inne r
(cytoplasmic) fac e o f th e membran e (Wooding , 1971a , 1977 ; Freudenstei n
et al., 1979) . Droplet s d o no t contac t th e plasm a membran e direcdy , bu t
rather thi s material . Whic h constituent s o f thi s electron-dens e materia l
recognize an d interac t wit h constituent s o n th e drople t surfac e remain s t o
be elucidated. Immunomicroscopi c (Frank e ^â/., 1981 ; Jarash ^â/., 1981 )
and biochemica l studie s (Freudenstei n et ai, 1979 ; Mathe r an d Keenan ,
1983; Nier a an d Mather , 1990 ) hav e show n butyrophili n an d xanthin e
oxidase, tw o prominen t protein s associate d wit h th e MLGM , t o b e majo r
constituents of the electron-dense materia l on the cytoplasmic face of apical
plasma membrane. Butyrophilin , a hydrophobic transmembran e glycopro -
tein, i s highl y concentrate d a t th e apica l surfac e o f milk-secretin g cell s
(Franke et ai, 1981 ; Niera an d Mather , 1990) . Xanthin e oxidas e i s distrib-
uted throughou t th e cytoplasm , bu t appear s t o b e enriche d a t th e apica l
cell surfac e (Jarasc h et ai, 1981) . Butyrophilin , whic h i s acylated (Keena n
et ai, 1982 ) an d bind s phospholipid s tightl y (Freudenstei n et aL, 1979) ,
has bee n believe d t o b e involve d i n mediatin g interactio n betwee n lipi d
droplets and apica l plasm a membrane . Recently , th e gen e fo r butyrophili n
was clone d an d sequence d (Jac k an d Mather , 1990) . Fro m th e inferre d
primary amin o aci d sequence , i t wa s no t apparen t ho w butyrophili n
could interac t wit h lipi d droplets . Sinc e butyrophili n ha s a n exoplasmi c
N-terminus an d a singl e membrane-spannin g domain , interactio n wit h
lipid droplets must occur with the 257-residue C-termina l domain , if in fact
this protei n doe s interac t wit h lipi d droplets . Fro m th e primar y sequence ,
the C-termina l domai n ha s n o obviou s hydrophobi c regions , bu t hydro -
phobic domain s coul d resul t fro m acylatio n o f serine-threonin e residues .
One possibilit y fo r interactio n o f butyrophili n i s wit h protein s o f th e lipi d
droplet surface rathe r than with lipids (Jack and Mather , 1990) . This could
be with proteins of the lipid droplet surface directly , or through complexe s
formed wit h cytoplasmic proteins. Since butyrophilin an d xanthine oxidas e
show a propensit y t o associat e wit h eac h other , a butyrophilin—xanthin e
oxidase comple x coul d b e involve d i n lipi d drople t interaction . Th e func -
tion tha t xanthin e oxidas e ma y play i n th e recognitio n o r envelopmen t
process remain s obscure .
10 Thoma s W . Keena n and Stuart Patto n
IV. Natur e an d Frequency of Cytoplasmi c

Crescents
A smal l compartmen t o f mil k tha t ha s receive d limite d attentio n i s cres -

cents o f cytoplas m o n mil k fa t globules . Thi s trul y i s a unique componen t
because technicall y crescent s ar e par t o f th e mamma l tha t mad e th e mil k
rather tha n a tru e secretor y produc t o f th e gland . Thus , crescent s mak e
their ow n distinctiv e contributio n t o th e compositio n an d propertie s o f
milk.
A. Mode of Crescent Formation
In th e proces s o f thei r secretion , mil k fa t globule s usuall y ar e envelope d

smoothly an d compactl y b y apica l membran e o f th e lactatin g cell . I n th e
electron microscope , th e droplet of fa t undergoin g secretio n appear s t o be
in clos e associatio n wit h membran e ove r it s entir e surface . However , o n
occasion, th e closur e o f th e membran e behin d th e projectin g fa t drople t
appears to occur by a route through th e apical cytoplasm rathe r than alon g
the drople t surface . Th e resul t i s that a fat globul e i s secreted wit h a piec e
of cytoplasm , a so-called crescen t o r signet, attached . Thes e ca n var y fro m
thin slivers of cellular material to situations in which the crescent dominate s
the globule . A typical huma n globul e wit h crescen t i s show n i n Figur e 1 .
A secon d mechanis m b y which crescent s ma y for m ha s been propose d
by Wooding (1977). The apical region of the lactating cell is populated wit h
secretory vesicle s containin g th e ski m phas e o f milk , whic h i s secrete d b y
exocytosis. Woodin g propose d tha t o n occasio n thes e vesicle s gathe r be -
hind a fat droplet that is in the proces s of secretio n an d trap cytoplasm int o
secretion wit h th e droplet . I t i s no t know n whethe r on e o r th e othe r o f
these mechanism s predominate . On e migh t expec t th e entrapmen t mech -
anism t o occasionally produc e membrane-boun d vesicle s of cytoplas m fre e
of a fa t droplet .
On th e assumptio n tha t som e significan t proportio n o f crescent s o n
milk fa t globule s for m a s a resul t o f a closure o f apica l plasm a membran e
through cytoplas m behin d th e fa t droplet , Husto n an d Patto n (1990 )
suggested tha t a n abnormalit y i n th e protei n coat , repute d t o bin d th e
membrane t o th e fa t drople t (Frank e et a/. , 1981) , i s responsible . Th e
principal protein s o f thi s coa t ar e butyrophili n an d xanthin e oxidas e
(Freudenstein et ai, 1979) . Inadequat e productio n o r distributio n o f thi s
coat comple x migh t interfer e wit h adhesio n o f th e membran e t o th e
droplet. Butyrophili n i s reported t o be acylate d wit h long-chai n fatt y acid s
(Keenan et a/., 1982) . Variatio n i n thi s acylatio n als o ma y alte r th e surfac e
properties o f th e protei n an d it s functio n a s a coa t (adhesive ) substance .
Determining wha t cause s crescen t formatio n a t th e molecula r leve l seem s
very challenging . Fo r example , th e phenomeno n appear s t o b e subjec t t o
2. Th e Structur e o f Mil k 1 1
Figure I Electro n micrograp h o f a typica l huma n mil k fa t globul e bearin g a crescen t o f

cytoplasm. Not e arra y o f well-preserve d roug h endoplasmi c reticulu m (rER ) in cytoplasm an d
membrane (arrow , lowe r left ) surroundin g lipi d drople t (L ) and cytoplasm . Scal e ba r denote s
0.5 îm .
diurnal variatio n i n th e human , wit h greates t number s o f crescent s bein g

formed i n th e evenin g (Patto n an d Huston , 1988) . The protein s an d fa t o f
milk ar e als o subjec t t o diurna l variations . Thi s make s possibl e a situatio n
in whic h synthesi s an d secretio n o f mil k fa t mayb e favore d a t a tim e whe n
structural element s neede d fo r globul e secretio n ar e i n shor t supply . I t i s
also possibl e tha t natur e favor s th e productio n o f crescen t materia l fo r
some benefi t i t has i n th e nurslin g (discusse d furthe r unde r Sectio n IV , D) .
B. Properties of Cytoplasmic Crescents
Cytoplasmic crescent s hav e bee n observe d t o contai n al l th e variou s mem -

branes an d organelle s o f th e lactatin g cel l excep t th e nucleus . A s a result ,
they tak e u p fluorescent dyes , such a s acridine orange , i n th e sam e manne r
as d o cells . Thi s i s a usefu l propert y i n quantifyin g crescent s (se e follow -
ing). I t is said tha t th e age of cells is revealed b y the fluorescent hue , orang e
being youn g an d yello w bein g older . Suc h variation s ca n b e see n i n
crescents. Usin g acridin e stainin g t o visualize crescents , Patto n an d Husto n
(1988) observe d thei r gradua l disappearanc e ove r 3 6 h r i n huma n mil k
stored a t 4°C . Thi s mean s tha t som e substance s originall y associate d wit h
milk fa t globule s progressivel y becom e par t o f th e ski m milk . Mil k accu -
mulates i n th e mammar y glan d followin g it s secretio n fro m th e cell , an d
removal o f mil k b y nursin g o r machin e i s neve r complete . Thus , prio r t o
milk removal , deterioratio n o f crescent s ca n procee d i n th e gland . Th e

difference i n ag e o f crescent s ca n b e detecte d i n thi n section s o f plastic -
embedded mil k viewed i n th e electron microscope . "Fresh" crescents loo k
like fresh specimen s of tissue; the ER is laminar and well-defined. I n olde r
crescents, the ER is open, rounded, and swollen looking but with ribosomes
still attached. Still later, ER is not recognizable as such. The cytoplasm then
has a totally disorganized, granular appearance (Patton and Huston , 1988) .
Globule populations with a high proportion of crescents exhibit a more
complex patter n o f protein s b y SDS-PAGE tha n d o low-crescent popula -
tions (Patto n an d Huston , 1988) . Presumably, th e man y additiona l mino r
bands tha t ar e eviden t aris e fro m th e cytoplasmi c component s i n th e
crescent. I t should b e possible to identify som e of these minor protein s by
immunostaining o f Wester n blots .
When membran e i s released fro m huma n mil k fa t globule s by churn -
ing, on e ca n isolat e a crescent-ric h fractio n b y low-spee d centrifugatio n
(1200g- fo r 1 0 min ) o f th e buttermilk . Viewin g o f thi s fractio n i n th e
electron microscop e reveal s that , despit e al l th e manipulatio n o f th e
sample, the shapes of crescents are preserved, suggesting that the structur e
of cel l cytoplas m i s no t amorphou s bu t somewha t define d (Patto n an d
Huston,-^ 1988), mos t probabl y b y element s o f th e cytoskeleto n (Schliwa ,
1986).
C. Detectio n and Quantification of Crescents
Jannsen an d Walstr a (1982 ) originated us e of the fluorescent dye , acridine

orange, for stainin g crescents. They als o devised a method o f quantifyin g
the amount of crescent material in milk based on enumeration o f crescents
in a defined volum e of milk and calculating their mass. Patton and Husto n
(1988) utilize d thi s procedur e fo r stainin g an d viewin g globule s wit h
crescents bu t pursue d dat a o n th e basi s o f wha t proportio n o f globule s
contain crescents. Both kinds of data are useful, e.g. , the Jannsen-Walstra
approach emphasize s how relatively small the amount o f crescent materia l
is in milk s of th e variou s specie s an d ho w littl e lactatin g cellula r materia l
is being lost from th e gland, and the Patton-Huston evaluatio n yields some
insight into globules with crescents as a proportion o f secretio n events . I n
one sampl e o f huma n milk , Patto n an d Husto n (1988 ) foun d tha t ther e
were almos t a s man y globule s wit h crescent s (44% ) a s without . Bot h
approaches suffe r th e limitations o f ligh t microscop y wit h respec t t o
viewing ver y smal l globules . However , contro l experiment s (Patto n an d
Huston, 1988 ) to assess crescents on such globules ( < 3 jim) indicated tha t
they accoun t fo r abou t 12 % o f th e tota l crescents . Sinc e smal l globule s
make u p 8 0 t o 90 % o f th e population , thi s implie s tha t ther e i s a muc h
stronger tendenc y fo r cresent s t o for m durin g secretio n o f th e large r
globules. Whil e i t i s als o possibl e t o enumerat e crescent s usin g electro n
microscopy (Patto n an d Huston , 1988) , ther e ar e limitations wit h thi s
approach. The mil k must be given structure with agarose or other suitabl e
agents; a t highe r magnifications , th e globule s nee d t o b e concentrate d

somehow s o that ther e ar e a sufficient numbe r o f the m i n th e field o f view .
In doin g thi s ther e i s a dange r tha t th e populatio n selecte d wil l no t b e
representative an d tha t crescent s ma y b e destroye d i n th e process .
Crescents hav e bee n identifie d i n associatio n wit h th e mil k fa t globule s
of al l specie s examine d t o date . T h e proportio n o f globule s wit h crescent s
varies betwee n an d withi n species . Woodin g et al. (1970 ) estimate d tha t
1-5% o f the globules in goa t and guine a pi g milk had crescents , while cow's
milk ha d relativel y fe w (abou t 1%) . Jansse n an d Walstr a (1982) , i n a
comparative study , measure d crescen t quantit y i n milk s o f severa l species ,
including cow , goat , rat , pig , sheep , rabbit , an d human , an d reporte d co w
to hav e th e leas t (7. 1 mg/k g milk ) an d rabbi t t o hav e th e mos t (13 1 mg/k g
milk). I n a stud y o f 5 0 huma n mil k donor s (Husto n an d Patton , 1990) ,
incidence o f crescent s o n fa t globule s range d fro m 1 to 29% . Mos t (80% )
fell betwee n 3 and 10 % and th e mea n (±SD ) wa s 7. 2 ± 4.2% . Two poole d
bovine mil k samples , both representin g ove r 10 0 animals, contained 1 % o r
less o f fa t globule s wit h crescents . T h e apparen t evolutionar y persistenc e
of cytoplasmic crescent s on huma n mil k fa t globule s suggests that the y ma y
have beneficia l effect s i n th e young .
In th e cours e o f thei r stud y o f 5 0 lactatin g women , Husto n an d Patto n
noted tha t diurna l an d geneti c factor s seeme d t o b e involve d i n crescen t
production. Ther e wa s definite evidenc e tha t evenin g sample s o f mil k ha d
higher crescen t number s tha n thos e collecte d i n th e morning . Tw o sister s
consistently showe d muc h highe r level s o f globule s wit h crescent s ( 2 5 -
44%) tha n other s i n th e study . Moreover , thi s characteristi c persiste d
during thei r followin g lactation . On e possibl e hypothesi s i s tha t thes e
sisters hav e onl y on e cop y o f th e butyrophili n gene . Frequen t milkin g an d
administration o f oxytoci n hav e bee n reporte d t o favo r secretio n o f glob -
ules wit h crescent s (Wooding , 1977) . Such treatment s woul d ten d t o mak e
for lactatin g cell s wit h cytoplas m distende d int o th e alveola r lume n alon g
with projectin g fa t droplets , a configuratio n tha t migh t facilitat e crescen t
formation.
In th e Huston-Patto n study , a numbe r o f factor s tha t di d no t see m t o
be involve d i n th e crescen t phenomeno n wer e ag e o f th e donor , stag e o f
lactation, whic h breast , volum e o f mil k expressed , a particula r fractio n
during a complete expressio n o f a breast, and mil k lipid o r protei n content .
They als o compare d crescen t incidenc e i n dair y cow s an d bee f cow s an d
found n o difference , whic h suggest s tha t selectio n fo r mil k productio n i n
cattle ma y no t b e a relevan t variable .
D. Significance of Crescent s
It ma y see m unlikel y tha t somethin g a s quantitativel y limite d a s crescent s

of cytoplas m o n mil k fa t globule s ca n hav e muc h significance . However , i t
has t o be remembere d tha t th e newbor n i s receiving mil k aroun d th e cloc k
day afte r day . Further , i t i s no w wel l appreciate d tha t man y biologicall y
14 Thoma s W. Keena n and Stuaut Patto n
important molecule s ar e effectiv e a t ver y lo w level s includin g enzymes ,

hormones, receptors in and on cells, growth factors , and trac e elements. I n
addition, crescent s ca n b e viewe d a s a sourc e o f foreig n antigen s (th e
mother's) tha t ma y hav e som e conditionin g effec t o n th e developin g
immune syste m o f th e newborn .
Even i f crescent s hav e n o nutritiona l value , ther e ar e a numbe r o f
other significan t consideration s concernin g them . A n understandin g o f
crescent formation a t the molecular level will do much t o further clarify th e
mechanism o f milk fat globule secretion . The implicatio n tha t butyrophili n
is essential t o th e latte r an d ma y b e insufficien t a t times, thereb y resultin g
in crescen t formation , need s research . I n th e preparatio n o f mil k fa t
globule membran e b y churnin g o r freeze-thaw , crescent s ten d t o concen -
trate in the membrane factio n an d constitute a significant impurit y i n som e
species. Method s whic h involv e a further purificatio n step , suc h a s centri -
fuging i n a density gradient , ma y eliminate thi s problem. Th e discover y o f
heretofore unreporte d biologica l molecule s i n milk raises such questions as
where ar e the y i n th e milk? , i.e. , i n wha t compartmen t an d ho w di d the y
gain entry ? Answerin g th e forme r ca n hel p t o defin e th e latter . Crescent s
represent a n importan t rout e o f cellular , a s oppose d t o secretory , sub -
stances int o milk . I n tha t connection , crescent s represen t a mean s o f
sampling specificall y th e lactatin g cel l withou t havin g t o biops y th e anima l
and isolat e th e particula r cell s fro m thei r tissu e matrix . Thi s i s a n impor -
tant consideratio n wit h th e human . Fo r thi s purpose , huma n mil k fa t
globules ca n b e isolate d (Patto n an d Huston , 1986) , churned , th e butte r
removed, an d th e buttermil k use d a s a suspension solutio n o f cytoplasmi c
constituents fro m th e lactatin g cell . O f course , thi s syste m wil l contai n
MLGM, bu t substance s originatin g fro m th e cytoplasm , suc h a s mRNAs ,
can b e readil y distinguishe d fro m thi s "contaminant. "
V. Siz e and Membrane Are a Distributio n of Mil k

Lipid Globules
A. Globule Size
Milk lipi d globule s o f specie s examine d t o date fal l int o thre e overlappin g
size distributions : smal l wit h diameter s centere d belo w 1 îm, intermediat e
with diameter s i n th e 3 t o 5 [A m range, an d larg e globule s wit h a mea n
diameter o f abou t 8 t o 1 0 [im . Som e o f th e latte r approac h 2 0 [i m an d i t
is fel t tha t thi s group , fo r th e mos t part , i s forme d b y postsecretio n
merging o f globules . Thus , mil k fa t globule s tha t ar e mos t typica l a s
secretory product s belon g t o th e tw o smalle r groups . However , a surpris -
ingly larg e proportio n o f th e tota l globul e population , 7 0 t o 90 % i n th e
bovine an d human , lie s i n th e first grou p belo w 1 |im i n diameter . Whil e
these globule s mus t accoun t fo r mos t o f th e secretor y events , the y contai n

< 5 % o f th e tota l mil k lipid . Globule s o f th e large-diamete r grou p mak e
up a very smal l par t o f th e tota l globul e population , estimate d t o be 0.01%
in th e human , bu t represen t 1 t o 4 % o f th e lipid . Therefore , th e inter -
mediate group , comprisin g roughl y 1 0 t o 30 % o f th e globul e numbers ,
accounts fo r 90 % o r mor e o f th e tota l lipid . Thes e findings ar e fro m
studies b y Walstr a (1969 ) an d Rueg g an d Blan c (1981) .
The averag e diamete r o f mil k lipi d globule s o f specie s examine d (cow ,
human, buffalo , goat , ewe , sow ) i s i n th e rang e o f 3 t o 5 îm . However ,
there ar e som e complication s i n arrivin g a t precis e figures fo r mea n
diameter. T h e population s o f smal l globules , < 1 |i m i n diameter , ar e
beyond enumeratin g limit s possibl e wit h th e ligh t microscop e o r Coulte r
counter. A s a consequence , thei r number s an d size s ofte n hav e bee n
ignored. Tw o studie s employin g th e Coulte r counter , on e o f th e co w
(Walstra, 1969 ) an d th e othe r fo r th e huma n (Riieg g an d Blanc , 1981) ,
have derive d value s fo r smal l globule s b y extrapolatio n wit h lo w relativ e
uncertainty. Usin g thi s approach , Walstr a determine d th e volume/surfac e
average diamete r (d^^) fo r Jerse y an d Fresie n cow s t o b e 4. 5 an d 3.3 4 îm ,
respectively. Usin g simila r methodology , Riieg g an d Blan c calculate d rfvs
for matur e huma n mil k t o b e 4. 0 îm . Fo r detail s o f th e theor y an d pro -
cedures use d i n thes e studies , th e origina l literatur e shoul d b e consulted .
It woul d b e interestin g t o kno w a t what , i f any , lowe r diamete r th e
globule populatio n fall s of f sharply . Thi s woul d indicat e whethe r ther e i s
a siz e belo w whic h fre e intracellula r lipi d droplet s d o no t exis t o r globul e
secretion fro m th e cel l doe s no t occur . Resolutio n o f thi s proble m wil l
require a n electro n microscop y approach . Presumably , th e larg e numbe r
of ver y smal l lipi d globule s i n mil k ar e secrete d b y th e sam e mechanis m
and carr y th e sam e membran e a s large r globule s (Deene y et al., 1985) .
Globule siz e range s i n alveola r lumen s wer e observe d t o b e th e sam e a s
those fo r lipi d droplet s insid e lactatin g cells (Wooding, 1971a) . Stemberge r
and Patto n (1981 ) als o observe d tha t th e lipi d drople t population s i n
lactating tissu e o f cow , cat , rabbit , rat , an d mous e wer e compose d o f tw o
groups, simila r t o thos e i n th e milk , i.e. , smalle r one s < 1. 5 |i m an d
intermediate i n th e 1. 5 t o 8 î m range . N o evidenc e o f ver y larg e droplet s
was take n b y the m t o indicat e tha t th e latte r mus t resul t fro m postsecre -
tation fusio n o f smalle r globules .
B. Membrane Surface Area
Riiegg and Blan c (1981 ) estimat e tha t th e lipi d globul e surfac e are a i n 1 ml
of matur e huma n mil k i s 500 cm^ . I n a liter o f milk , thi s would b e 500,00 0
cm^ o f surfac e o r roughl y th e floor spac e i n a roo m 2 3 ft^ . Whethe r on e
thinks o f mil k i n term s o f processin g an d storag e effect s o r digestio n an d
behavior i n th e gut , thi s i s a larg e amoun t o f surface . W e ca n assum e tha t
the surfac e are a o f lipi d globule s i n mil k i s roughl y equivalen t t o tha t o f
globule membran e (i.e. , one sid e o f tha t membrane) . However , ther e is , as

mentioned i n th e sectio n o n membran e stabilit y (Sectio n VII , A) , th e
question o f ho w muc h membran e ha s sluffe d fro m th e globule s int o th e
skim milk . Thi s wil l vary , a s wil l th e proportio n o f (small ) globule s lef t i n
the ski m mil k b y centrifugal separation . S o it is not possibl e t o deriv e ver y
precise estimate s o f th e membran e surfac e are a i n ski m milk . Usin g suc h
criteria a s th e amoun t o f membran e protein s an d activitie s o f membran e
enzymes, i t appear s tha t abou t one-thir d t o one-hal f o f th e membran e
materials in milk is recovered i n the skim milk. I t seems reasonable t o think
of thi s a s surfac e are a lik e tha t o n globules , althoug h i t ma y represen t
various configuration s an d eve n b e membran e sheet s wit h bot h exo - an d
endoplasmic face s exposed . Th e membran e materia l i n ski m mil k ofte n i s
referred t o as the "fluff" fraction becaus e i t is a loose layer that rests on th e
casein pelle t followin g centrifugation . Fo r furthe r characteristic s o f thi s
fraction, especiall y it s appearanc e i n th e electro n microscope , se e Stewar t
etal, (1972) .
V I . Natur e o f th e Mil k Lipi d Globul e Membran e
A. Isolation of Milk Lipid Globule Membrane
The membran e surroundin g lipi d globule s i n mil k closel y resemble s

plasma membran e i n ultrastructur e i n tha t i t has a typical bilaye r appear -
ance, wit h th e spac e betwee n bilayer s bein g comparabl e t o tha t o f plasm a
membrane, an d ha s a n externall y dispose d glycocaly x (Moni s et ai, 1975 ;
Freudenstein et ai, 1979 ; Sasak i an d Keenan , 1979 ; Frank e et a/., 1981) .
This membran e i s characterized , a s ar e differentiate d region s o f apica l
plasma membrane , b y th e appearanc e o f th e electron-dens e materia l
associated wit h th e inne r fac e o f th e membran e (Figur e 2 ) (Wooding ,
1971b; Freudenstei n et a/., 1979 ; Frank e et a/., 1981) . Som e o f th e plasm a
membrane initiall y surroundin g globule s ma y b e los t followin g secretion ,
within alveola r lumina , o r i n expresse d mil k (Wooding , 1974) , bu t esti -
mates o f th e exten t o f thi s los s var y widel y (reviewe d i n Mathe r an d
Keenan, 1983) . I n region s o f globule s wher e th e bilaye r membran e ap -
pears t o hav e bee n lost , a granula r materia l cover s th e surface . Thi s
material ma y b e th e coatin g whic h wa s o n th e surfac e o f lipi d droplet s
within milk-secretin g cells . Stabilit y o f MLG M i s discusse d i n mor e detai l
under Sectio n VII .
Membranes ca n b e release d fro m mil k lipi d globul e suspension s b y
several processes , includin g freezin g an d thawing , vigorou s agitatio n
(churning) (Keena n et al., 1988) , exposur e t o nonioni c detergent s lik e
Triton X-10 0 (Patton , 1982 ) o r t o conjugate d bil e salt s lik e taurodeoxy -
cholate (Patto n et ai, 1986) , o r b y suspensio n i n polar , aproti c solvent s
(Dapper et a/. , 1987) . Afte r release , membranou s materia l normall y i s
Figure 2 Electro n micrograp h o f a co w MLG M preparation . Thi s preparatio n consist s

largely o f sheet s o f membran e whic h displa y typica l bilaye r membran e structure . T h e inne r
(originally cytoplasmic ) fac e o f th e membran e i s coate d wit h a densel y stainin g materia l o f
variable thickness. The coa t materia l o n th e inner fac e o f th e membran e largel y is amorphous,
but regularl y arrange d particulat e o r globula r structur e sometime s i s observed (insert) . Scal e
bar denote s 0. 2 îm . This plat e wa s reproduce d fro m Keena n et al. (1977 ) wit h permissio n o f
the publisher .
collected b y centrifugation a t g force s u p t o or exceedin g 100,000 . Alter -

natively, membran e materia l ca n b e cause d t o aggregat e b y reducin g th e
pH or by adding ammonium sulfate to the suspension, and can be collected
by low-spee d centrifugatio n (Brunner , 1965 ; McPherso n an d Kitchen ,
1983). Mil k lipi d globule s an d membrane s derive d therefro m ar e identica l

or nearl y s o i n distributio n o f phospholipids , bu t ther e ar e majo r quanti -
tative difference s i n polypeptid e compositio n o f intac t globule s an d iso -
lated MLG M (Mathe r an d Keenan , 1975 ; Keena n etai, 1988) . Dependin g
upon th e metho d an d temperatur e o f globul e disruption , appreciabl e
amounts o f protein s an d pola r lipid s remai n associate d wit h th e congeale d
lipid (butter ) o r ar e disperse d i n th e aqueou s phas e entraine d i n th e
congealed lipid . When suspension s o f washe d lipi d globule s ar e churne d a t
low temperatures , a considerabl e amoun t o f proteinaceou s materia l re -
mains associate d wit h th e surface s o f congeale d lipi d droplet s (Deene y et
al.y 1985). The metho d an d exten t o f washin g o f lipi d globule s t o remov e
milk seru m constituents , prio r t o releas e o f th e membrane , als o ca n alte r
composition o f th e materia l recovere d ultimatel y a s MLGM . T h e exten t o f
removal o f periphera l (extrinsic ) protein s unde r variou s washin g condi -
tions ha s no t bee n quantified . T h e metho d use d fo r collectio n o f MLGM ,
and an y subsequen t step s use d t o remov e entraine d material s fro m th e
membrane, ca n alte r compositio n a s well . Relativel y larg e proportion s o f
xanthine oxidas e an d som e membran e glycoprotein s ca n b e selectivel y
removed fro m MLG M (Mathe r et ai, 1977 ; Keena n et al, 1977a) .
B. Gross Composition
Over 95 % of th e tota l lipid s i n mil k fro m cow s (Huan g an d Kuksis , 1967 ;

Jenness, 1974 ; Patto n an d Jensen , 1976 ) an d human s (Bracc o et a/., 1972 ;
Jensen et ai, 1980 ; Blanc , 1981 ; Jensen, 1989 ) i s recovere d i n th e globul e
fraction upo n centrifuga l fractionatio n o f mil k into globule and mil k seru m
or ski m mil k phases , an d 95 % or mor e o f th e globul e lipid s ar e triacylgly -
cerols. Muc h o f th e lipi d i n mil k seru m i s presen t i n a heterogeneou s
fraction o f membran e an d membran e fragment s which , fro m th e mil k o f
cows, ha s bee n characterize d thoroughl y (reviewe d i n Patto n an d Keenan ,
1975; Keena n et al, 1988) . Phospholipid s (3 0 t o 45% ) an d triacylglycerol s
(40 t o 55% ) compris e th e bul k o f th e lipid s i n mil k seru m (Huan g an d
Kuksis, 1967 ; Patto n an d Keenan , 1971 ; Patto n et al, 1973 , 1980b) . T h e
percentage o f th e tota l globul e mas s accounte d fo r b y membran e materia l
has no t bee n determine d wit h certainty . Fro m dat a summarize d b y Brun -
ner (1965 ) an d b y Mulde r an d Walstr a (1974) , membrane-associate d ma -
terials may comprise fro m abou t 2 to more tha n 6 % of the mas s of globules .
This rang e o f value s mus t b e considere d a s a crud e estimat e a t best , a s
differences i n globul e an d membran e preparatio n method s hav e a majo r
effect o n th e valu e obtained . Globule s mus t b e washe d sufficientl y t o re -
move adsorbed o r adheren t mil k seru m constituents , ye t extensive washin g
also wil l remov e loosel y associate d bu t tru e constituent s o f th e membrane .
An additiona l complicatio n i s that membran e materia l wil l be entraine d i n
the congeale d lipid s (butter ) whe n globule s ar e treate d t o releas e MLG M
material at temperatures below the solidification poin t of the triacylglycero l

mass.
Available informatio n o n gros s compositio n o f MLG M fro m co w an d
human milks is provided i n Table I . Compositional dat a for human MLG M
largely i s fro m a limite d numbe r o f studies . I n mos t analyses , protein s
plus lipid s togethe r hav e accounte d fo r ove r 90 % o f th e membran e dr y
weight, bu t relativ e proportion s o f protein s an d lipid s var y widely . Thi s
variation ca n be du e t o differences i n method s fo r releas e an d recover y o f
membrane an d t o othe r factor s suc h a s breed, age , an d stag e o f lactation .
Once isolated , MLG M ca n b e subfractionate d b y isopycni c densit y gradi -
ent centrifugatio n int o a rang e o f densit y classes . Densit y o f fraction s i s
correlated inversel y wit h bot h phospholipi d an d tota l lipi d contents . Al l
fractions hav e simila r polypeptid e patterns , a s judged fro m pattern s ob -
served upo n separatio n o f polypeptide s b y electrophoresi s unde r dena -
turing conditions . I n on e study , variou s densit y subfraction s als o wer e
found t o hav e simila r o r identica l specifi c activitie s (unit s o f activity/uni t
protein) o f certai n MLGM-associate d enzyme s (Mathe r et a/., 1977) . How -
ever, Kitche n (1977) , usin g a different procedur e t o prepar e an d subfrac -
tionate MLG M accordin g t o density , note d larg e difference s i n enzym e
specific activitie s i n differen t densit y classes .
The greates t variatio n i n membran e compositio n i s th e conten t o f
neutral lipids , principall y triacylglycerols . Wha t par t o f th e triacylglycero l
associated with isolated MLG M represents a true membrane constituent, i n
contrast t o an adsorbed o r entrained contaminant , i s not known . Sinc e cel l
TABLE I
Gross Composition of Cow an d Human Mill < Lipid Globule Membrane Preparation s
Constituent clas s Unit Cow'' Human*
Protein weight % 25 t o 6 0 —
Total lipid s mg/mg protei n 0.5 t o 1. 1 1.46
Phospholipids mg/mg protei n 0.13 t o 0.3 4 0.35
Neutral lipid s mg/mg protei n 0.25 t o 0.8 8 1.1
Glycosphingolipids'^ [ig/mg protei n 13 32
Hexoses Âg/mg protei n 108 45
Hexosamines Hg/mg protei n 66 44
Sialic acid s |Ag/mg protei n 20 18
Glycosaminoglycans Hg/mg protei n 0.1 -
RNA Hg/mg protei n 20 15
''Taken fro m compilatio n i n Keena n et al. (1988) .

*Data fro m huma n MLG M compile d fro m Marte l et al. (1973) , Bouhour s an d Bouhour s
(1979), Takamizaw a et al (1986b) , an d Jensen (1989) .
'^Calculated fro m publishe d dat a assumin g averag e molecula r weight s o f neutra l glycosphin -
golipids an d ganglioside s o f 85 0 an d 1470 , respectively .
surface membranes , isolated fro m homogenate s o f mammar y glan d as well

as other tissues, contain only small amounts of triacylglycerols, one assume s
that th e triacylglycerol s associate d wit h MLG M i n par t originat e fro m th e
core lipid . O n a protein basis th e phospholipi d conten t o f MLG M i s mor e
constant tha n i s th e neutra l lipi d content ; a n averag e valu e fo r phospho -
lipid i s abou t 0.2 5 m g pe r milligra m protei n fo r th e cow , an d a simila r
value wa s obtained fo r human . Marte l et al. (1973 ) reporte d a much lowe r
value fo r phospholipi d (0.08 5 mg/m g protein ) fo r huma n MLGM , bu t
this valu e undoubtedl y i s erroneousl y low . MLGM , lik e plasm a mem -
branes, i s enriche d i n glycosphingolipid s i n relationshi p t o intracellula r
membrane systems . Th e tota l amoun t o f glycosphingolipid s (comprisin g
neutral glycosphingolipid s an d gangliosides ) i n cow MLG M i s about 1 3 \ig
per milligra m protein . Bouhour s an d Bouhour s (1979 ) reporte d a tota l
neutral glycosphingolipid conten t of human MLG M of 11. 5 M^g/mg protein.
The gangliosid e content of huma n MLG M has not been measured directly ,
but Otnaes s et al, (1983 ) reporte d a content o f 1 1 mg/ 1 milk, an d Takam -
izawa et al. (1986 ) foun d 1 0 t o 2 3 ^mo l o f gangliosid e siali c aci d pe r lite r
of huma n milk . Usin g averag e value s fo r mas s of MLG M i n milk , an d fo r
mass o f MLG M whic h i s protein, th e approximat e amoun t o f ganglioside s
would b e abou t 2 0 jAg/m g protein . Severa l author s hav e foun d RN A i n
MLGM preparations ; Swop e an d Brunne r (1965 ) mad e carefu l measure -
ments and foun d abou t 2 0 ^ g RNA/mg protei n i n cow MLGM , and Marte l
et al. (1973 ) foun d 1 5 jig RNA/mg protei n i n human MLGM . Extractio n o f
membranes with high ionic strength buffers reduce d RN A levels in MLGM
to about 1 0 ^g per milligra m protei n (Jarasc h et al., 1977) . This RN A ma y
be a constituent o f th e primar y MLGM ; alteratively, i t may originat e fro m
ribosomes associate d wit h th e surface s o f lipi d droplet s withi n th e cel l
(Dylewski et al., 1984 ) or from entrainment o f E R membranes or ribosome s
in cytoplasmic crescents . DN A ha s not been detecte d i n cow (Jarasc h et al.,
1977) an d huma n (Marte l et al, 1973 ) MLG M preparations . Hexos e
(glucose, galactose , mannose , an d fucose) , hexosamin e (glucosamin e an d
galactosamine), an d siali c aci d (Ts^-acetyl - an d N-glycoylneuramini c acids )
contents of MLG M have been measure d (Tabl e I) . In aggregate total , thes e
carbohydrates amoun t t o just under 0. 2 m g pe r milligram protein . Mos t of
the MLGM-associate d carbohydrate s ar e covalentl y boun d t o protein s an d
lipids; i t i s no t likel y tha t muc h fre e carbohydrat e i s associate d wit h th e
MLGM. Glycosaminoglycans , normall y associate d wit h basemen t mem -
branes, hav e bee n isolate d fro m co w an d huma n MLG M preparations . Li s
and Moni s (1978 ) identifie d hyaluroni c acid , chondroiti n sulfate , an d
heparin sulfat e i n th e glycosaminoglyca n fractio n fro m co w MLGM . How -
ever, th e valu e o f 5 8 î g glycosaminoglycans pe r milligra m protein , calcu -
lated fro m th e dat a o f Li s an d Moni s (1978) , seem s t o b e unrealisticall y
high. Shimiz u et al. (1981 ) confirme d th e presenc e o f hepari n sulfat e an d
chondroitin sulfat e i n glycosaminoglycans fro m MLGM ; they foun d a total
glycosaminoglycan conten t o f abou t 0. 1 |A g per milligra m protei n i n co w
MLGM an d a 5 - t o 10-fol d highe r amoun t i n huma n MLGM .
C. Lipid Composition
1. Neutral Lipids
Values reporte d fo r th e amount s o f mos t o f th e lipi d classe s o f huma n
MLGM fal l withi n th e rang e o f value s reporte d fo r co w MLG M (Tabl e II) .
In membrane s fro m bot h sources , triacylglycerol s ar e th e mos t abundan t
lipid class . Since preparativ e metho d ha s a majo r influenc e o n th e amoun t
of triacylglycero l associate d wit h MLGM , i t i s likel y tha t som e o f th e
triacylglycerols originat e fro m th e cor e lipid s an d adsor b ont o o r partitio n
into th e membran e material . Fatt y aci d compositio n o f MLGM-associate d
triacylglycerols differ s fro m tha t o f mil k fa t i n tha t MLG M triacylgly -
cerols contai n considerabl y highe r proportion s o f long-chain , saturate d
fatty acid s (principall y palmitat e an d stearate ) (Kitchen , 1977) . Vasi c an d
DeMan (1966 ) an d Bracc o et al. (1972 ) foun d tha t whe n fa t globule s wer e
destabilized a t temperature s abov e 37°C , isolated MLG M wa s no t enriche d
in high-meltin g triacylglycerols . Walstr a (1974 ) suggeste d tha t thes e high -
melting triacylglycerol s ma y b e derive d fro m fa t crystal s whic h "contami -
nate'* th e membran e durin g th e coolin g an d churnin g process . Result s
from microelectrophoreti c characterizatio n o f lipi d globule s le d Newma n
and Harriso n (1973 ) t o conclud e tha t th e oute r surfac e o f th e MLG M
contains littl e neutra l lipid . Trac e t o substantia l quantitie s o f mono - an d
diacylglycerols usuall y ar e foun d i n MLG M lipids . Whethe r thes e partia l
glycericdes ar e tru e constituent s o f membrane s o r ar e product s o f lipolyti c
degradation o f triacylglycerol s o r phosphoglyceride s i s no t known . T h e
amounts o f unesterifie d fatt y acid s foun d i n MLG M preparations , a t leas t
from cow , var y widely . Thi s variatio n ma y b e du e t o variatio n i n lipolyti c
activity. Sterol s an d stero l ester s invariabl y ar e foun d i n MLG M lipids ;
however, ther e i s extensive variatio n i n stero l conten t judged fro m value s
reported fo r co w MLGM . Som e o f thi s variatio n ma y b e th e resul t o f
preparative method-induce d differentia l partitionin g o f sterol s betwee n
core an d membran e lipid s (discusse d i n Keena n et a/., 1988) . Cholestero l
is presen t i n lipi d droplet s befor e secretio n a s mil k lipi d globules , bu t it s
distribution betwee n th e cor e lipid s an d materia l o f th e surfac e coa t i s
unknown (Dylewsk i et al., 1984) . Cholesterol i s a known an d abundan t lipi d
constituent o f plasm a membrane , fro m mammar y glan d (Keena n et ai,
1970; Kann o et ai, 1987) , a s wel l a s othe r tissue s (reviewe d i n Va n Meer ,
1989). An ultrastructura l approach , i n whic h cholesterol-filipi n complexe s
were visualize d i n freeze-fractur e replicas , provide d evidenc e fo r th e
presence o f cholestero l bot h i n o r a t th e surfac e o f cor e lipids , an d i n th e
MLGM i n intac t mil k lipi d globule s (Martin , 1989) . This observatio n doe s
not rul e ou t partitionin g o f cholesterol betwee n membran e an d cor e lipid s
before globule s ar e harveste d an d destabilized . I n MLG M fro m bot h cow s
and humans , stero l ester s accoun t fo r a smal l proportion , 10 % o r less , o f
the total sterols. Fat globules, but no t necessaril y MLGM , from huma n mil k
contain a muc h highe r amoun t o f cholestero l tha n d o thos e i n co w mil k
22 Thomas W. Keena n and Stuart Patto n
TABLE I I
Lipid Composition of Cow an d Human Mill c Lipid Globule Membrane Preparation s
Constituent clas s CoW Human *
% of total lipi d
Triacylglycerols 62 5 8
Diacylglycerols 98
Monoacylglycerols Trace 0. 6
Sterols 0.2 t o 2 0. 7
Sterol ester s 0.1 t o 0. 3 Trac e
Unesterified fatt y acid s 0.6 t o 6 7. 3
Hydrocarbons 1.2 Trac e
Phospholipids 26 t o 3 1 2 3
% of tota l phospholipi d
Sphingomyelin 22 2 6
Phosphatidyl cholin e 36 3 0
Phosphatidyl ethanolamin e 27 3 7
Phosphatidyl inosito l 11 5
Phosphatidyl serin e 41
Lysophosphatidyl cholin e 22
''As compiled i n Keena n et al. (1988); Patton and Keena n (1975).

*As compiled i n Jensen (1989) .
(Braco et ai, 1972) . Cholesterol i s the major sterol in human an d cow milks,
accounting fo r ove r 90 % o f th e tota l stero l fraction . Abou t 1 7 differen t
sterols have been isolate d fro m co w milk ; thos e whic h hav e been identifie d
include 7-dehydrocholesterol , campesterol , stigmasterol , an d ^-sitostero l
(reviewed i n Blanc, 1979) . I n addition t o cholesterol, 7-dehydrocholestero l
and phytosterol s hav e bee n foun d i n huma n milk . Whic h o f thes e sterol s
are i n MLG M ha s ye t t o b e determine d wit h eithe r species . Squalene , th e
abundant hydrocarbon o f cow and human mil k fat (Bracco etai, 1972) , has
been identifie d a s a constituen t o f co w MLG M (Thompso n et al, 1961) .
P-Carotene als o i s presen t i n th e hydrocarbo n fractio n o f co w MLG M
(Thompson et a/., 1961) , but mos t of th e carotenoid o f th e globul e appear s
to b e i n th e cor e lipi d (Patto n et al., 1980) . Carotene s als o ar e associate d
with huma n mil k lipi d globules , bu t th e distributio n betwee n cor e lipid s
and th e MLG M ha s ye t t o b e determined .
2. Phospholipids
Phospholipids o f mil k ar e mainl y presen t i n lipi d globule s (abou t 60%

of th e total ) an d i n th e heterogeneou s membran e fractio n o f ski m mil k
(about 40% ) (Huan g an d Kuksis , 1967 ; Patto n an d Keenan , 1971) . Th e

phospholipids o f lipi d globule s ar e mainly , i f no t exclusively , recovere d
with MLG M whe n globule s ar e destabilize d a t temperature s o f 40°C . Th e
same five majo r phospholipids , with a simila r patter n o f distribution , ar e
present i n MLG M fro m co w an d huma n (Tabl e II) , a s wel l a s i n mil k o r
MLGM fro m ass , camel, India n buffalo , pi g (Morriso n 1968) , goa t (Patto n
and Keenan , 1971) , mous e (Calberg-Bac q et aL, 1976) , an d ra t (Keena n et
ai, 1971) . Huma n MLG M ha s relativel y les s phosphatidy l choline , bu t
relatively mor e phosphatidy l ethanolamin e an d sphingomyelin , tha n doe s
cow MLGM . Phosphatidy l serin e an d phosphatidy l inosito l accoun t fo r a
higher proportion o f phospholipid s i n cow MLG M than i n human MLGM ,
but thi s differenc e wa s no t observe d upo n compariso n o f phospholipi d
distribution i n whol e mil k specimen s fro m thes e specie s (Morrison , 1968) .
The phospholipi d distributio n patter n see n wit h MLG M i s simila r t o tha t
found wit h plasma membrane s fro m mammar y glan d (reviewe d i n Keena n
et a/., 1988 ; Kanno , 1990 ) an d othe r organ s i n tha t sphingomyeli n i s hig h
and phosphatidy l cholin e i s low . Intracellula r membrane s hav e a muc h
lower sphingomyeli n t o phosphatidy l cholin e rati o tha n tha t foun d with
plasma membrane s an d MLGM . I n additio n t o th e five majo r phospho -
lipids, lyso-derivative s o f th e majo r phospholipid s als o ar e foun d i n
MLGM, bu t thes e ar e relativel y mino r constituent s i n fres h sample s han -
dled s o a s t o minimiz e lipolyti c activity . Wha t proportion s o f th e variou s
phosphoglyceride classe s ar e alky l o r alkeny l ether s i s no t know n a s
analytical method s use d t o establis h distributio n o f phospholipid s wer e
unable t o mak e suc h separations . Alky l an d alkeny l ether s wer e foun d i n
choline an d ethanolamin e phosphoglycerid e fraction s fro m whol e mil k o f
cows (Ha y an d Morrison , 1971) .
Several worker s hav e note d extensiv e similarit y i n distributio n o f
phospholipids o f ski m milk , o r membrane s isolate d therefrom , an d lipi d
globules an d i n distributio n o f majo r fatt y acid s withi n eac h phospholipi d
class. Given this apparent unit y of mil k phospholipids , i t has been assume d
that thos e o f whol e mil k an d MLG M originat e fro m a commo n cellula r
source and , b y inference , ar e identica l o r nearl y s o i n fatt y aci d composi -
tion. Actua l dat a t o validat e thi s assumptio n ar e lacking . A ver y larg e
number o f mino r fatt y acid s hav e bee n identifie d i n mil k lipids , bu t w e
have n o informatio n o n th e distributio n o f mos t o f thes e fatt y acid s withi n
individual lipi d classe s an d canno t assum e tha t ther e i s no t a preferentia l
occurrence o f an y give n mino r fatt y aci d i n a particula r lipi d clas s o f
MLGM o r o f ski m milk .
3. Glycosphingolipids
Glycosphingolipids, relativel y mino r constituents o f th e MLG M (Tabl e
I), hav e bee n th e subjec t o f a numbe r o f investigation s ove r th e pas t sev -
eral years . Thi s attentio n ha s bee n du e t o th e recognitio n tha t certai n
glycosphingolipids an d product s o f glycosphingolipi d catabolis m hav e
functional role s i n a numbe r o f biologica l phenomena , suc h a s cell-cel l

interaction, differentiation , proliferation , immun e recognition , transmem -
brane signaling , an d a s receptor s fo r certai n hormones , growt h factors ,
and toxin s (reviewe d i n Hakomori , 1981 , 1990 ; Fishman , 1986 ; Hannu n
and Bell , 1989 ; Karlsson , 1989) . Two genera l classe s of glycosphingolipid s
have bee n foun d i n mil k an d MLGM : neutra l glycosphingolipid s (wit h
uncharged sugars , commonl y calle d cerebrosides ) an d acidi c glycosphin -
golipids (containin g siali c aci d an d calle d gangliosides) . Thes e lipid s hav e
a ceramid e ( a sphingosin e bas e t o whic h a fatt y aci d i s attache d vi a a n
amide bond ) t o whic h i s linked , throug h th e ceramid e primar y hydroxy !
group, a mono- or oligosaccharide. Co w MLGM contains two major neutra l
glycosphingolipids, glucosyl - an d lactosylceramides , i n nearl y equimola r
proportions. Neutra l glycosphingolipid s wit h mor e comple x carbohydrat e
structures hav e no t ye t been foun d i n co w MLGM . Glucosyl - an d lactosyl -
ceramides of MLG M are enriched i n long-chain fatt y acids, especially 16:0 ,
18:0, 22:0 , 23:0 , 24:0 , an d 24:1 , bu t appea r t o lac k hydroxylate d fatt y
acids. Thes e lipid s als o occur i n ski m milk , bu t thos e fro m ski m mil k hav e
relatively lesse r amounts o f th e 22- , 23-, and 24-carbo n fatt y acid s (Kayser
and Patton , 1970) . I n huma n MLGM , ther e i s a tota l neutra l glycosphin -
golipid conten t o f abou t 1 1 |ig/m g protei n (Bouhour s an d Bouhours ,
1979). I n huma n MLGM , ther e i s about doubl e th e amoun t o f monohex -
osylceramides a s dihexosylceramides. Galactosylceramid e comprise s abou t
88%, and glycosylceramide abou t 12% , of the total monohexosylceramides .
The dihexosylceramid e o f huma n MLG M i s lactosylceramid e (Bouhour s
and Bouhours , 1979) . Glucosyl - an d lactosylceramide s o f huma n MLG M
did no t contain hydroxylate d fatt y acids , but galactosylceramid e containe d
both hydroxylate d an d nonhydroxylate d fatt y acids . Amon g nonhydroxy -
lated fatt y acids , 16:0 , 22:0 , 24:0 , an d 24: 1 wer e mos t abundant , account -
ing fo r abou t 70 % o f th e tota l fatt y acid s i n neutra l glycosphingolipids .
Over 80 % of th e hydrox y fatt y aci d wa s accounte d fo r a s 22:0 , 23:0 , an d
24:0.
Nine differen t ganglioside s hav e bee n detecte d an d characterize d
structurally, a t leas t partially , i n co w MLG M o r buttermilk . Thos e identi -
fied t o dat e hav e bee n GM3 , GMg , GM, , GD3 , GDg , GDj b (Huang , 1973 ;
Keenan, 1974 ; Bushwa y an d Keenan , 1978) , GT3 , an d nove l branched -
chain mono - an d trisialoganglioside s (Takamizaw a et a/., 1986a) . I n mos t
investigations, GD 3 emerged a s th e majo r gangliosid e o f co w MLGM , an d
GM3 wa s th e nex t mos t abundan t gangliosid e homolog . Th e aggregat e
total o f th e othe r ganglioside s amounte d t o perhap s n o mor e tha n 20 % of
the total ganglioside content of the membrane. GD3 and GM3 are the majo r
gangliosides o f huma n MLGM ; trac e amount s o f ganglioside s tentativel y
identified a s GM g an d GM j als o hav e bee n detecte d i n huma n MLG M
(Takamizawa etal, 1986b ; Laegreid an d Otnaess , 1986) . I n human MLG M
there i s a majo r differenc e i n gangliosid e distributio n wit h stag e o f lacta -
tion (Takamizaw a et ai, 1986b) . I n mil k sample s collecte d 2 - 6 day s
postpartum, th e GM3:GD 3 rati o wa s 0. 2 t o 0.3 , bu t i n sample s collecte d
60-390 day s postpartum , thi s rati o wa s greate r tha n 3 . Mil k collecte d a t

8-40 day s postpartu m ha d GMgiGD s ratio s intermediat e betwee n thes e
extremes. Ganglioside s o f MLG M hav e fatt y aci d composition s simila r t o
those o f neutra l glycosphingolipid s an d sphingomyelin , perhap s indicatin g
that al l o f thes e sphingosine-containin g lipid s originat e fro m a commo n
pool o f ceramides .
4, Fat-Soluble Vitamins and the Membrane
Certain o f th e vitamins . A, D , E, and K are sai d t o be fa t soluble . Whe n

foods, suc h a s mil k an d mil k products , ar e extracte d wit h so-calle d fa t
solvents, thes e vitamin s ar e obtaine d i n th e solvent s alon g wit h th e lipids .
From this , on e migh t conclud e tha t thes e vitamin s i n th e nativ e stat e ar e
dissolved i n th e triacylglycero l cor e o f th e mil k lipi d globule . Whil e thi s
may b e tru e o f este r forms , suc h a s o f vitamin s A an d E , i t woul d no t b e
true o f thos e containin g pola r groups , an d al l fou r vitamin s exis t a t leas t
in som e proportio n i n form s containin g unesterifie d hydroxy l groups .
Under physiologica l conditions , thes e latte r form s ar e hydrate d an d woul d
resist solutio n i n lipi d droplets . Mor e likely , the y occu r i n membrane s
including th e MLGM , an d ar e oriente d i n th e surfac e o f lipi d droplets .
However, hea t connecte d wit h foo d processin g (pasteurizatio n an d steril -
ization) migh t overcom e thes e distribution s an d actuall y extrac t som e o f
the fat-solubl e vitamin s int o lipi d droplets .
There ar e suggestion s i n th e literatur e tha t on e o r mor e o f th e
fat-soluble vitamin s o f mil k resid e i n par t i n th e MLG M an d ar e accounte d
for mor e adequatel y o n a surfac e distributio n basi s rathe r tha n o n fa t
content. Ther e i s also the possibilit y tha t thes e vitamin s ar e associate d wit h
carrier protein s an d membran e materia l i n th e ski m milk . Bu t th e dat a ar e
far to o sketch y a t thi s tim e t o enabl e presentatio n o f concentration s o f th e
vitamins, eithe r i n th e MLG M o r i n th e intac t globule , o f eithe r huma n o r
cow. To achiev e tha t goa l wil l require systemati c fractionatio n an d analysi s
of mil k an d it s compartments . A t th e moment , th e bes t guidanc e i s fro m
the dat a o n fat-solubl e vitami n concentration s i n whol e milk .
Related t o th e forgoin g consideratio n i s th e questio n o f carotenoids .
Some o f these , principall y a - an d p-carotene , ar e precursor s o f vitami n A .
While ther e ar e report s o f carotenoid s i n th e MLG M (Thompso n et ai,
1961), the y apparend y d o no t occu r ther e wit h consistenc y (Brunner ,
1965). I n a stud y o f th e distributio n o f carotenoid s i n bovin e mammar y
tissue an d mil k lipi d globule s (Patto n et ai, 1980a) , MLG M wa s foun d t o
be devoi d o f carotenoids .
D. Protein Composition
Consideration i s given her e mainl y t o protein s o f th e huma n an d co w mil k

lipid globule s becaus e the y accoun t fo r mos t o f th e informatio n an d the y
are o f greates t interes t an d significance . A t th e sam e time , i t shoul d b e
borne i n min d tha t th e majo r huma n an d co w globul e proteins , suc h a s

butyrophilin an d xanthin e oxidase , see m t o sho w commonalit y acros s
species. Thi s suggest s tha t whil e thes e protein s hav e bee n evolvin g amon g
species t o yiel d som e variation s i n size , sequence , etc. , the y retai n proper -
ties essentia l t o thei r function s i n th e lactatin g cel l an d th e nursling .
Protein represent s onl y abou t 1 % o f th e globul e mas s o r 0. 3 t o 0. 4
g/liter o f huma n o r bovin e mil k (Patto n an d Huston , 1986) . Fe w o f th e
individual protein s hav e been isolate d an d extensivel y characterized . How -
ever, thei r behavio r an d stainin g propertie s o n SDS-gel s giv e usefu l
indications o f thei r concentrations , relativ e molecula r weight s (Mr's) , an d
whether o r no t the y contai n carbohydrate . Resolutio n o f protein s fo r th e
two specie s b y SDS-PAG E i s show n i n Figur e 3 . Th e principa l band s ar e
identified bu t ther e ar e man y mino r band s tha t remai n unknown . Th e
number o f thes e wil l depend o n th e siz e of sampl e an d stainin g technique .
No doubt , som e ar e enzyme s (Sectio n VI , E) . Wester n blottin g couple d
with immunostainin g afford s a ver y sensitiv e techniqu e fo r investigatin g
the identitie s o f thes e bands .
1. Compartmentation of Globule Proteins

The questio n arise s a s t o whic h protein s o f th e globul e ar e associate d
with the membrane, whic h with the original fa t droplet surface , an d whic h
with th e intervenin g cytoplasmi c space . Becaus e o f th e potentia l fo r pro -
ducing artifact s i n isolating thes e compartments, thes e questions canno t b e
answered with certainty. I n fact, the process of globule secretion ma y effec t
change i n the positio n o f th e proteins . I f one use s the evidence o f protein s
released b y globules subjecte d t o churning t o identify thos e residin g i n th e
cytoplasmic space, two principal bands are seen on gels . One a t M^ 155,000
corresponding t o xanthin e oxidas e an d th e othe r a t M^ 15,000 (Patto n et
al.y 1986) . Sinc e xanthin e oxidas e i s a component o f th e coa t comple x o n
the cytoplasmi c (inner ) fac e o f th e globul e membrane , i t is reasonable tha t
some o f i t migh t b e release d b y churning . Exceptin g th e contributio n o f
proteins i n cytoplasmi c crescents , a s discusse d unde r Sectio n IV , th e
secretory mechanis m effectivel y expel s th e cytoplas m fro m mos t globule s
and one woul d no t expect muc h contributio n t o total globul e protei n fro m
this compartment . A compariso n o f th e butte r phas e fro m th e churnin g
process wit h th e isolate d MLG M reveal s essentiall y identica l ge l protei n
patterns (Patto n et ai, 1986) . Thi s suggest s tha t th e protein s o f intac t
globules are largely components of th e MLGM. However , ther e is evidence
that lipi d droplet s isolate d fro m withi n th e lactatin g cel l carr y a spectru m
of protein s originatin g i n th e endoplasmi c reticulu m (Deene y et al, 1985) .
Carryover o f thes e protein s t o th e secrete d globule s i s discusse d unde r
Section II .
2. The Principal Globule Proteins

Resolution o f th e principal globul e protein s by SDS-PAGE i s shown i n
Fig. 3. Note the relatively strong staining of the bands for xanthine oxidas e
HB
7 » - .... .
Figure 3 A n SD S ge l comparin g protein s o f huma n (H ) an d bovin e (B ) mil k lipi d globules .

Major matchin g band s fo r th e tw o specie s ar e indicate d b y arrowheads . Relativ e molecula r
weights (Mr ) i n kD a ar e a t right . T h e numbere d position s a t lef t correspon d to : 1 , xanthin e
oxidase (monomer) ; 2 , PAS-I V (Af ^ 8 0 kDa) ; 3 , butyrophilin; 4 , butyrophilin-relate d protei n
(Mr 62. 5 kDa) ; 5 , PAS-V I (glycoprotei n B) ; 6 , actin-kerati n (? ) band; 7 , component 21 . T h e
bracket (right ) locate s tw o contaminatin g casei n bands . T h e sample s eac h containe d 5 0 \ig o f
protein. T h e ge l (12.5 % acrylamide ) wa s staine d wit h Coomassi e blue .
and butyrophilin i n both the human an d th e cow samples. This is generally

the cas e i n othe r specie s a s well . Thes e relativel y hig h concentrations ,
coupled wit h th e occurrenc e o f th e tw o protein s acros s specie s lines ,
suggest a rol e o f fundamenta l importanc e fo r the m i n th e MLGM . I n
comparing th e pattern s fo r th e huma n an d co w samples , i t i s evident tha t
the forme r ha s man y mor e mino r bands . Thes e ar e contributed a t least i n
part by cytoplasmic crescents (see Section I V and Figur e 1) . The mucin s are
not observed i n Figur e 3 because o f thei r limite d penetratio n int o a 12.5 %
acrylamide ge l an d thei r poo r stainin g wit h Coomassi e blue . However ,
see Figur e 4 fo r th e bovin e mucin . Availabl e informatio n o n th e globul e
proteins i s summarize d belo w an d i n Tabl e III .
a. Mucin(s) . Hig h molecula r weigh t glycoproteins , no w know n a s ep -

ithelial mucins , have been foun d i n milk lipid globules of all species studie d
to date . I n th e huma n ther e ar e actuall y tw o suc h protein s detectabl e b y
SDS-PAGE; on e tha t resolve s i n th e stackin g ge l (3 % acrylamide) an d th e
other tha t enters a 4% acrylamide runnin g gel . Thes e protein s hav e man y
names and abbreviations . They ar e designated her e a s mucin A and muci n
B, respectively . Th e forme r i s approximatel y 80 % an d th e latte r abou t
50% carbohydrate. Bot h ar e very hig h molecula r weigh t ( > 400,000) . Co w
globules only exhibit one muci n composed o f about 50% carbohydrate an d
ranging i n M^ from 170,00 0 t o 205,000. W e refe r t o this mucin a s periodi c
acid-Schiff reagen t I (PAS-I ) i n accor d wit h th e nomenclatur e o f Mathe r
[ ^m ^ ^ .._ , -20 5
-** s i
^ ^ ^ WKM H ^ ^ ^ W JNHM K ^ ^ ^ ^ T
i
"^ -11 - 9 76
n H I 11 .u
M1 mi ^^ ^"1 -66
^ ^ ^ SJM ^ <ll««'^"''' » *'•'«?»» '
mm flK M&^ Mt t
Figure 4 A n SDS gel showing polymorphism o f the bovine milk lipid globule mucin, PAS-I,
among fiv e animal s (i n brackets) . Not e variabl e numbe r an d mobilit y o f bands . Positio n o f
molecular weight references i s indicated in kDa at left. The gel contained 6 % acrylamide and
was stained with silver reagents. Each of the five samples were 1 0 jig of total globule protein.
and Keena n (1975 ) fo r th e co w MLG M proteins . I t appear s t o b e o f th e

same famil y o f glycoprotein s a s huma n muci n B . Ther e i s considerabl e
variation i n th e M^ of th e mil k lipi d globul e mucin s amon g species . A
further uniqu e aspect of these glycoproteins is their polymorphism (Patto n
and Huston , 1987 ; Swallo w et ai, 1987 ) whic h result s fro m variabl e
numbers o f a tandeml y repeate d 20-amin o aci d segmen t (Gendle r et ai,
1988; Spice r et ai, 1991) . Thus, the individual alleles , one fro m th e mothe r
and on e fro m th e father , usuall y expres s protein s o f differen t sizes . Thi s
is manifest on SDS-gels by two bands which vary in mobility from one mil k
sample t o another . Bot h th e stackin g ge l an d runnin g ge l mucin s exhibi t
this typ e o f polymorphis m (Patto n an d Huston , 1987 ; Patto n et al, 1989) .
However, som e species , suc h a s th e cow , goat , an d mouse , hav e onl y th e
running ge l mucin , an d i n th e cas e o f th e mouse , th e polymorphis m
appears to have been lost although mutated tandem repeats remain (Spice r
etal, 1991) . The polymorphis m o f co w PAS-I , a s revealed b y SDS~PAGE ,
is shown in Figure 4. Under the conditions in Figure 4, the human B muci n
would be at the very top of th e gel. Characterizationa l wor k has been don e
on huma n muci n A (Shimiz u an d Yamauchi , 1982 ) an d B (Shimiz u et a/. ,
1986) an d o n co w PAS- I (Sno w et a/., 1977) . Huma n muci n B (Gendle r et
ai, 1990 ) an d th e mous e muci n tha t resolve s i n th e runnin g ge l o n
SDS-PAGE (Spice r et ai, 1991 ) hav e bee n sequence d vi a th e cDNAs . Th e
20-amino aci d repeatin g uni t is rich in serines and threonines , whic h serv e
as O-glycosylatio n sites . O n SDS-gels , th e mucin s stai n readil y wit h PAS ,
but ver y weakl y i f a t al l wit h Coomassi e blu e (CB) .
b. Xanthin e oxidase (monomer , 155,000 ; band I of Figur e 3) . O n gels , thi s

protein stain s strongl y wit h C B bu t doe s no t stai n wit h PAS . Thus , i t
contains littl e o r n o carbohydrate . However , ther e i s a huma n
carbohydrate-containing protei n whic h occurs in the 155,00 0 M^-region o f
the ge l whic h ha s bee n isolate d an d characterize d regardin g amin o aci d
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and carbohydrat e compositio n (Ima m et al, 1981) . I n th e nativ e state ,

xanthine oxidas e exist s a s a homodimer , molecula r weigh t 283,000 . Th e
Mr observed o n gel s fo r th e monome r o f 155,00 0 i s thu s somewha t high .
Its properties as an enzyme and its unique composition ar e discussed unde r
Section VI , E . Th e mos t perplexin g aspec t o f thi s membran e componen t
concerns it s functio n i n MLG M o r i n lipi d globul e secretio n (discusse d
below).
c. Butyrophili n (ban d 3 o f Figur e 3) . Thi s protein , with M^ of 66,000 ,

stains bot h wit h C B an d PAS . I t i s th e majo r protei n componen t o f th e
MLGM bu t i t seem s likel y tha t xanthin e oxidas e ma y a t time s constitut e
nearly a s muc h o f th e tota l globul e protein . I n th e isolatio n o f th e
membrane fro m th e globul e a considerable amoun t o f xanthin e oxidas e i s
shed becaus e i t i s no t a tru e integra l membran e component , bu t i s com -
plexed rathe r loosely , a t leas t i n part , wit h butyrophili n i n th e membran e
coat A butyrophilin-lik e protei n ha s bee n detecte d i n MLG M o f a variet y
of othe r specie s (Hei d et aL, 1983) . Sequencin g an d othe r evidenc e indi -
cates tha t cow butyrophili n i s composed o f a n extracellula r domai n whic h
is glycosylated , a transmembran e region , an d a cytoplasmic tai l (Jac k an d
Mather, 1990) . A glycoprotei n o f M ^ 70,000 isolate d fro m huma n MLG M
(Imam et aL, 1981 ; Cerian i et aL, 1983 ) appear s t o b e butyrophilin . It s
amino acid and carbohydrat e compositio n hav e been determine d (Ima m et
aL, 1981 ) an d monoclona l antibodie s t o i t hav e bee n prepare d (Cerian i et
aL, 1983) .
d. M r 62,500 (band 4 of Figure 3) . Thi s protei n ma y be a close structura l

relative o f butyrophilin .
e. M, 46.00 0 t o 52,00 0 (ban d 5 o f Figur e 3 ; als o know n a s PAS-V I an d

glycoprotein B) . Thi s protei n stain s wit h bot h C B an d PAS . Th e huma n
protein appear s t o be somewha t smalle r a t M^ 46,000 tha n tha t of th e co w
(Mr 49,500) an d a similar goa t protei n i s eve n slighd y highe r (M ^ 52,000)
(Patton an d Hubert , 1983) . Al l thre e bin d th e lectin , concanavali n A . Th e
amino aci d an d carbohydrat e composition s o f th e co w protei n hav e bee n
determined (Basc h et aL, 1976) . Becaus e som e breas t tumor s expres s thi s
protein, monoclonal antibodies to it have been prepared and use d t o screen
for breas t cance r (Cerian i et aL, 1983) .
f. M^ 42,000 (ban d 6 of Figur e 3) . Althoug h th e ban d fo r thi s protei n i s

broad o n SDS-gels , i t doe s no t appea r t o contai n carbohydrate . Th e
molecular weigh t rang e i s tha t o f actin s an d som e keratins , whic h woul d
be plausibl e sinc e protein s o f th e filamentou s networ k ma y functio n i n
connecting th e membran e t o th e celF s cytoskeleton .
g. M r 39,000 . Ther e i s a relativel y broad , PAS-positiv e ban d i n thi s

region o n SDS-gels . Th e huma n glycoprotei n ha s bee n isolate d an d
partially characterized includin g amino acid and carbohydrate compositio n

(Imam et a/., 1982) . I t is not know n whethe r thi s protei n i s the sam e a s th e
HLA-DR-like antige n a t M, 35,00 0 detecte d b y Wima n et al (1979) .
h. AI 4 15,00 0 (ban d 7 o f Figur e 3) . Thi s ban d i s see n o n gel s resolvin g

human, cow, and goa t globule proteins . I t appears to be carbohydrate free .
Since i t i s release d readil y durin g churnin g o r freezing-thawin g o f glob -
ules, it is probably a peripheral rathe r tha n a n integral membran e protein ,
and i t ma y resid e o n th e inne r (cytoplasmic ) fac e o f th e membrane .
Suggested relationship s betwee n protein s o f th e huma n an d bovin e
globule mus t b e considere d tentativ e unti l furthe r evidenc e i s provide d
such a s cross-reactivit y with antibodie s o r homolog y o f amin o aci d se -
quences. Ther e i s stil l muc h characterizationa l wor k t o b e don e o n th e
globule proteins . Fo r example, th e bovin e protei n i n th e M^ 54,00 0 regio n
(Figure 3 ) seem s t o hav e receive d n o attentio n t o date , no r ha s th e M^
15,000 componen t eviden t i n th e pattern s o f bot h specie s (ban d 7 ,
Figure 3) .
3. Lectin Binding
Lectins hav e bee n use d extensivel y i n th e characterizatio n an d purifi -

cation o f MLG M proteins . A stud y b y Farra r et al. (1980 ) i s particularl y
useful i n tha t bindin g o f 1 2 differen t lectin s t o intac t huma n an d co w
globules wa s evaluated . Anothe r informativ e stud y o n bindin g o f lectin s
to MLG M protein s i s tha t o f Murra y et al. (1979) . Peanu t lectin , whic h
binds t o |3-D-galactopyranosyl-(l,3)-N-acetyl-galactosamine , wa s use d t o
purify huma n muci n B (Shimizu an d Yamauchi , 1982) . The bovin e mucin ,
PAS-I, als o bind s peanu t lecti n strongl y (Patto n et al., 1989) . Ima m et al.
(1981) employe d concanavali n A in the isolatio n o f thei r M^ 70,000 huma n
glycoprotein (butyrophili n ?) ; the goa t glycoprotei n wit h M^ 52,000 bind s
concanavalin A strongl y (Patto n an d Hubert , 1983) .
4. Functions of the Milk Lipid Globule Membrane and Its Proteins

The protein s o f milk , includin g thos e o f th e lipi d globules , ca n b e
viewed a s having function s i n th e mammar y glan d o f th e mothe r o r in he r
nursling. Further , i t i s conceivabl e tha t a particula r mil k protei n migh t
have function s i n bot h locations . I n thi s regard , i t i s helpfu l t o vie w mil k
in a n evolutionar y sense . Biologically , mil k i s a ver y crucia l elemen t i n
survival o f th e mammalia n species . Exclusiv e o f man , wit h hi s capacity fo r
sophisticated manipulatio n o f foo d requirements , n o mil k mean s deat h
of th e newborn . Limite d o r poo r qualit y mil k mean s undernourishe d
and sickl y youn g wh o ma y no t matur e o r reproduce . N o doub t ther e ha s
been selectio n o f mil k protein s t o ensur e adequat e mil k qualit y an d
quantity, thu s perpetuatin g species . B y the sam e token , i n th e evolution o f
a mammal , i t i s possibl e tha t a mil k componen t tha t wa s indispensabl e a t
one tim e ma y hav e becom e nonessentia l a t a late r stage , an d i f harmless ,
carried alon g geneticall y a s th e specie s furthe r evolved . Thi s i s no t quit e

applicable t o a protein i n the sens e tha t proteins wil l have som e nutritiona l
value i n an y event . Bu t th e likelihoo d i s that most , i f no t all , mil k protein s
have been hone d b y evolution t o facilitate survival of the particula r species.
The nutritiv e valu e o f mil k fo r th e youn g i s fairl y straightforwar d i n
that a very larg e numbe r o f require d nutrient s an d thei r amount s ca n b e
specified; mil k o f a given species , i n th e main , meet s th e requirement s fo r
young o f tha t species . Bu t beyon d thi s ther e ar e man y othe r comple x
considerations whic h ar e no w comin g t o th e fore . Suc h thing s a s cel l
growth factors , hormones , enzymes , antibodies , an d immunogeni c sub -
stances i n mil k ma y als o benefi t th e newborn . I n addition , th e contro l o f
gut microflor a i s o f critica l importanc e t o healt h an d developmen t o f th e
young human . Bein g mino r i f no t trac e component s o f milk , th e MLG M
proteins mak e ver y littl e contribution t o the classic nutritiv e valu e o f milk ,
but there i s likelihood tha t they ma y benefit th e young in the same manne r
as som e o f thes e foregoin g factor s tha t ai d wel l being .
Because o f th e dynami c need s an d relativ e underdevelopmen t o f
metabolic system s in the newborn , th e question o f wha t happens t o milk i n
the youn g ha s becom e a matte r o r risin g interest . I t i s a difficul t are a o f
research becaus e o f it s invasiv e requirements . Th e infan t brai n double s i n
size i n th e first 6 month s postpartu m o n a n exclusiv e die t o f huma n milk .
A difficul t questio n i s what mil k components , i f any , ar e use d intac t in th e
synthesis o f brai n durin g thi s period ? I n connectio n wit h possibl e contri -
bution b y th e MLGM , i t i s pertinen t tha t th e infan t brai n i s a n extensiv e
system o f evolvin g membranes . Thi s researc h are a become s eve n mor e
challenging b y reports, such a s Lucas et al. (1992 ) an d other s cited therein ,
that human-milk-fe d infant s develo p greate r intelligenc e o n averag e tha n
do thos e fe d formula .
a. Th e MLGM . A componen t o f mil k tha t ha s bee n a n integra l struc -

ture i n th e lactatin g cell , a s i s th e cas e o f th e MLGM , obviousl y wil l hav e
had importan t function s i n tha t cell . Ther e i s evidence o f MLG M protei n
involvement i n enzymatic , receptor , transport , immunologic , an d milk -
secretory functions . Muc h o f thi s i s by inferenc e fro m th e compositio n o f
the membrane, it s close resemblanc e t o plasma membranes i n general, an d
what i s known abou t thei r functions . However , i t is firmly establishe d tha t
the MLG M play s th e centra l rol e i n th e secretio n o f mil k lipi d globules .
With respec t t o possibl e function s o f th e MLG M i n th e nursling , w e
propose tha t on e functio n o f thi s materia l i s t o serv e a s a deco y fo r
pathologic bacteri a an d thei r toxin s i n th e infan t gut . Lik e th e intestina l
mucosa, th e lactatin g cell s ar e epithelia l cell s an d thei r membrane s ar e
epithelial membranes . Fo r man y enteri c bacteri a o f th e typ e tha t caus e
sickness i n th e young , bindin g t o th e intestina l mucos a i s a n essentia l first
step t o infection . Th e sam e i s tru e o f th e diarrhea-producin g toxin s o f
the typ e elaborate d b y Vibrio cholerae an d Escherichia coli. To b e effective ,
they mus t first bind t o ganglioside i n the mucosa l membrane . I f a constant
supply o f extraneous epithelia l membran e materia l i s coursing throug h th e

lumen o f th e gut , th e chance s tha t thes e bacteri a an d thei r toxin s ma y b e
tied u p b y suc h membran e ar e favorable , thu s minimizin g bindin g t o an d
invasion o f th e intestina l mucosa . I t i s a well-know n fac t tha t breast-fe d
infants ar e muc h les s afflicted wit h diarrhea l disorder s tha n ar e thos e give n
formula. W e conten d tha t i n th e human , MLG M an d it s component s pla y
an importan t par t i n thi s difference .
b. Mucin(s) . T h e followin g observation s an d speculation s appl y par -

ticularly t o the huma n B mucin tha t enters a 4% running ge l in SDS-PAG E
and i s variously name d MUC-1 , PAS-0 , and th e B- C mucin , amon g others .
Comparison o f th e tande m repeat s fo r th e huma n an d mous e mucin s
reveals tha t position s o f th e serine s an d threonine s ar e conserved , wherea s
amino acid s i n othe r position s hav e undergon e extensiv e mutatio n (Spice r
et ai, 1991) . Thi s strongl y implie s tha t th e oligosaccharid e chain s ar e a n
important functiona l elemen t o f thes e mucins , sinc e i n thes e location s th e
serines an d threonine s ar e glycosylatio n sites . T h e fac t tha t ther e i s fa r
more muci n an d i t i s o f highe r molecula r weigh t i n huma n mil k lipi d
globules tha n i n thos e o f th e co w (Patto n et al, 1989 ) o r mous e (Spice r et
a/., 1991 ) suggest s tha t th e mucin s hav e evolve d t o b e o f greate r impor -
tance i n th e human . Th e oligosaccharid e chain s o f th e mucin s firs t ar e
exteriorized o n th e apica l surfac e o f th e lactatin g cell . A plausibl e functio n
for the m i n thi s locatio n i s to serv e a s a barrie r agains t invadin g (mastitic )
microorganism. I t i s also reporte d tha t i n thi s location , the y ar e connecte d
on th e cytoplasmi c sid e o f th e plasm a membran e t o element s o f th e
cytoskeleton (Parr y et ai, 1990) . I t ha s bee n suggeste d tha t th e mucin s
function i n the immunorecognition syste m (Patto n an d Huston , 1987) . Th e
human muci n tande m repeat s carr y bloo d grou p antigen s (Dio n et a/. ,
1990) an d B an d T cel l epitope s (Gendle r et aL, 1990) . The T cel l epitop e
is recognize d b y cytotoxi c T cell s (Barn d et aL, 1989) . Huma n muci n B
inhibits th e growt h o f BALB/ c 3T 3 cell s (Shimiz u et a/., 1990) . I t ha s bee n
suggested tha t th e huma n mucin s facilitat e digestio n o f mil k lipi d globule s
(Shimizu an d Yamauchi , 1982 ; Buchhei m et ai, 1988) .
c. Xanthin e oxidase . I n th e lactatin g cel l thi s protei n tend s t o concen -

trate o n th e cytoplasmi c fac e o f th e apica l plasm a membran e althoug h i t is
dispersed throughou t th e cytoplas m (Jarasc h et al., 1981) . I n th e apica l
position i t become s complexe d wit h butyrophili n i n wha t i s know n a s th e
protein coa t o f th e MLGM . Whe n a matur e mil k lipi d drople t reache s th e
apical membrane , th e coate d membran e surfac e commence s t o envelo p
the drople t i n th e secretio n process . I t i s reasone d tha t propertie s o f th e
coat bind th e membran e t o th e drople t (Keena n an d Dylewski , 1991) . Thi s
appears mor e likel y i n tha t th e coa t protein s ar e reporte d t o b e acylate d
with long-chai n fatt y acid s (Keena n et al., 1982 ) which woul d enhanc e thei r
hydrophobic attractio n t o lipi d droplets . Thus , assistin g i n th e secretio n o f
milk lipi d globule s appear s t o b e a plausibl e functio n o f xanthin e oxidase .
Xanthine oxidas e occur s i n remarkabl y hig h concentration s i n mil k

(approx 3 5 mg/liter) . I t i s the riches t know n sourc e o f th e enzyme . Doub t
is cast on it s functioning a s an enzyme i n that it is nearly, if not completely ,
inactive i n th e mil k o f som e species , e.g. , huma n an d goat . Molybdenu m
deficiency ma y explai n th e lac k o f activit y i n huma n xanthin e oxidase . A t
this time , ther e i s n o goo d evidenc e wh y th e lactatin g cel l o r th e newbor n
would requir e suc h hig h level s o f xanthin e oxidase . A nutritiona l rol e fo r
xanthine oxidas e a s a purveyo r t o th e youn g o f trac e element s (iro n an d
molybdenum), sulfur , an d intac t flavin-adenine-dinucleotide comple x i s
another possibility .
d. Butyrophilln . Thi s protei n wa s s o name d becaus e o f it s apparen t

affinity fo r th e mil k lipi d drople t a t secretio n (Frank e et ai, 1981) . Thi s
seems t o b e a well-accepte d workin g hypothesis , discusse d above , o n
functions o f th e xanthin e oxidase-butyrophili n coa t complex . Fro m it s
sequence (Jac k an d Mather , 1990) , butyrophili n appear s t o b e a classica l
integral membran e protei n wit h a singl e transmembran e regio n an d a
sizeable cytoplasmi c tail . Thi s tai l apparentl y i s a recepto r fo r o r i s inter -
active with xanthine oxidase. Butyrophilin doe s not appear to be expresse d
in any other tissue of the body and i t is only evident in and fro m mammar y
tissue durin g lactatio n (Jac k an d Mather , 1990) . Thi s make s i t likel y tha t
the rol e o f butyrophili n i s exclusivel y relate d t o lactation . Butyrophili n i s
glycosylated i n it s exoplasmi c segmen t an d possibl y i n it s cytoplasmi c tai l
(Jack an d Mather , 1990) . Th e function(s ) o f thi s glycosylatio n i s no t
known.
In addition , th e huma n globul e membran e contain s histocompatibilit y
antigens in the form o f tw o glycoproteins a t M^. 35,000 an d 28,00 0 (Wima n
et ai, 1979) . Ther e ar e n o know n function s fo r th e othe r majo r protein s
of th e mil k lipi d globul e liste d i n th e foregoing , i.e. , thos e a t M ^ 80,000,
46,000, 42,000 , 39,000 , an d 15,000 .
D. Enzyme s of Milk Lipid Globule Membranes
Over 2 5 enzymati c activitie s hav e bee n detecte d an d measure d i n MLG M

from co w mil k (Tabl e IV) . I n som e cases , thes e differen t activitie s ma y
be catalyze d b y th e sam e enzym e usin g differen t substrate s o r acceptors .
Well ove r hal f o f th e enzyme s detecte d i n co w MLG M ar e member s o f
the hydrolas e class . Oxidoreductase s ar e nex t i n abundance , followe d b y
transferases. Aldolase , th e onl y lyas e reported , i s a well-know n cytosoli c
enzyme, an d it s presenc e ma y b e indicativ e o f cytoplasmi c crescents .
Acetyl-CoA carboxylase , th e onl y ligas e reported , wa s presen t i n enzymat -
ically inactive for m (Shrive r et ai, 1989) . To date , isomerases hav e not been
reported a s constituent s o f MLG M preparations . Wit h huma n MLGM ,
about 1 1 differen t enzymi c activitie s hav e bee n reporte d (Tabl e IV) .
Several enzyme s wit h hig h specifi c activitie s i n MLGM , suc h a s adenosin e
2. Th e Structure o f Milk 3 5
TABLE IV
Enzymatic Activities Detecte d in Cow an d Human Mil k Lipi d Globule Membran e
Preparations
Enzyme" Cow* Human''
Alkaline phosphatas e 3.1.3.1 A

Acid phosphatas e 3.1.3.2 A
5'-Nucleotidase 3.1.3.5 A C
Phosphodiesterase I 3.1.4.1 A C
Inorganic pyrophosphatas e 3.6.1.1 A
Nucleotide pyrophosphatas e 3.6.1.9 A
Phosphatidic aci d phosphatas e 3.1.3.4 A
Adenosine triphosphatas e 3.6.1.3 A C
Cholinesterase 3.1.1.8 A
UDP-glycosyl hydrolase s 3.2.1._ A
Glucose-6-phosphatase 3.1.3.9 A C
Plasmin 3.4.21.7 A
P-Glucosidase 3.2.1.21 A
P-Galactosidase 3.2.1.23 A
Ribonuclease I 3.1.4.22 A C
Thiamine pyrophosphatas e 3.6.1.6 C
Lipoamide dehydrogenas e 1.6.4.3 A
Xanthine oxidas e 1.2.3.2 A D
Thiol oxidas e 1.8.3.2 A
NADH oxidas e 1.6.99.3 A E
NADPH oxidas e 1.6.99.1 A
Catalase 1.11.1.6 A
Y-Glutamyl transpeptidas e 2.3.2.1 A
Galactosyl transferas e 2.4.1._ A F,G
Glycosyl transferase s 2.4._._ H
Aldolase 4.1.2.13 A
Acetyl-CoA carboxylas e 6.4.1.2 B
''Common or trivial name of enzyme i s followed b y the Enzym e Commission (EC ) referenc e
number.
^Letter indicate s tha t enzym e ha s bee n reporte d i n MLG M o f tha t species , an d key s th e
reference: A , reviewe d i n Keena n et al. (1988) ; B , Shrive r et al. (1989 ) foun d acetyl-Co A
carboxylase to be present in enzymatically inaaive form; C, Martel-Pradal and Got (1972); D,
Zikakis^/fl/. (1976) ; E. Burder ^â/. (1978) ; F, Martel etal (1973) ; G, Martel and Got (1976a);
H, Parod i et al. (1984) foun d enzyme s o f synthesi s o f dolicho l monophosphomannos e an d
dolichol monophosphoglucose , a s well as those involved i n transfer of glycosy l residu e fro m
these dolicho l derivativ e t o dolichol diphosphooligosaccharides .
triphosphatase, phosphodiesteras e I , an d 5'-nudeotidase , ar e enriche d i n

plasma membranes fro m severa l tissue s and frequentl y ar e used a s marker
enzymes fo r plasm a membranes . A s discusse d unde r a previou s section ,
xanthine oxidase is an abundant protein associated with milk lipid globules,
and thi s enzyme ca n be i n th e oxidas e o r dehydrogenase for m (Nakamur a
and Yamazak i 1982 ; Chen g et ai, 1988) . A numbe r o f enzymi c activitie s
present i n MLG M preparation s ar e associate d wit h intracellula r mem -
branes suc h as , fo r example , Golg i apparatu s (glycosyltransferase s an d
thiamine pyrophosphatase) , endoplasmi c reticulu m (glucose-6-phos -
phatase, glycosyltransferase s usin g dolicho l a s acceptor) , lysosome s (aci d
phosphatase, glycosidases) , an d cytoplas m (aldolase) . I n mos t cases ,
whether thes e activitie s ar e constituent s o f th e MLG M prope r o r ar e
present in cytoplasmic material s entrained betwee n th e membran e an d th e
core lipi d ha s no t bee n determined . Nevertheless , thes e activitie s ar e
associated wit h mil k lipi d globules , irrespectiv e o f thei r precis e localizatio n
within the globule. Some activities , concentrated i n one cellular membrane ,
are als o presen t i n othe r locations . Fo r example NAD H oxidase s (NADH -
cytochrome c an d ferricyanid e reductases ) ar e enriche d i n endoplasmi c
reticulum, bu t als o ar e constituent s o f Golg i apparatu s an d plasm a mem -
branes (Jarasc h et aL, 1979) . I n co w an d huma n MLGM , Brude r et al.
(1978) foun d th e NAD H oxidas e system , t o b e linke d t o b 5 an d P42 0
cytochromes. Anothe r sourc e whic h ca n potentiall y contribut e enzyme s t o
milk lipi d globul e membran e preparation s i s cell s i n milk , whic h ma y b e
entrained wit h globule s durin g recover y o f crea m an d no t b e remove d b y
the washing procedures . Anderso n an d Cawsto n (1975 ; cf. als o Anderson ,
1977) hav e foun d leucocyte s entraine d i n mil k lipi d globule s t o b e th e
source of at least portions of certain enzymic activities measure d i n MLGM
preparations. Marte l an d Go t (1976 ) foun d tha t MLG M fro m huma n mil k
would incorporat e fre e amin o acids into proteins. This observation implie s
that severa l enzyme s an d protei n factor s necessar y fo r polypeptid e syn -
thesis are presen t i n human mil k lipi d globules , bu t as yet ther e hav e bee n
no report s o n individua l component s o f th e protei n syntheti c apparatu s i n
MLGM.
VII. Reorganizatio n of the Membran e Durin g

Storage and Processing
As migh t b e expecte d o f a comple x biologica l structure , th e MLG M i s

fragile an d i n a stat e o f flux. Ther e ar e man y way s i n whic h it s stabilit y
may be viewed fro m th e standpoints of physica l an d chemical change , suc h
as it s physica l organizatio n an d continuit y o n globules , it s compositio n
(proteins an d hpids) , activit y o f it s enzymes , bindin g phenomena , dena -
turation o f proteins , oxidatio n o f lipids , an d subtl e biochemica l changes .
There i s goo d evidenc e o f a qualitativ e natur e tha t chang e occurs , bu t

there i s a paucit y o f quantitativ e dat a a s t o ho w durabl e th e membran e i s
in variou s circumstances .
A. Milk in the Gland
Milk as it is removed fro m th e human breast or the cow's udder already ha s

a history. Tha t is , time ha s elapsed t o varying degrees sinc e secretion fro m
the lactatin g cell o f ever y lipi d globule , casei n micelle , an d lactos e o r othe r
molecule i n th e milking . A ver y smal l par t o f th e milkin g ca n b e ver y ol d
in that a fraction, often estimate d at 15-20 % of total milk in the cow udder ,
is left i n th e glan d a t each milking . Thi s residua l mil k i s occluded i n smal l
ducts and alveoli. Fo r experimental purposes , such as to obtain very freshl y
secreted milk , i t ca n b e largel y remove d b y a serie s o f short-interva l
milkings subsequen t t o a complete milking . Thes e milkin g manipulation s
stimulate additional oxytoci n release , which in turn forces the residual mil k
out o f th e fine apertures . Administratio n o f oxytoci n an d additiona l
milking i s als o use d t o achiev e th e sam e end . However , thi s residua l mil k
normally remain s behin d an d mixe s wit h th e ne w mil k tha t accumulate s
prior t o th e nex t milking . Agai n a t th e nex t milking , a certain proportio n
of th e mil k i n th e glan d i s lef t behind . Thus , agin g o f th e mil k an d
associated change s begi n i n th e glan d and , dependin g o n th e conditions ,
continue i n variou s way s afte r th e mil k ha s bee n expressed . Thes e ar e
cogent consideration s whe n so-calle d freshnes s o f mil k i s concerne d an d
they ar e relevan t t o th e natur e o f th e MLGM .
Wooding (1971 ) ha s conclude d fro m studie s wit h th e electro n micro -
scope tha t th e MLG M i s unstable , an d soo n afte r secretio n o f th e globul e
from th e cel l i t loses membran e b y vesiculation int o th e ski m milk . O n th e
other hand , secrete d globule s exhibi t th e characteristi c structur e o f a
biomembrane o n thei r surface , an d membran e isolate d fro m mil k fa t
globules show s thi s structure a s well (Figur e 2) . Unfortunately , ther e i s n o
way o f seein g thi s membran e othe r tha n b y chemica l fixation, embeddin g
in plastic and thi n sectioning , followe d b y very high-magnificatio n viewing .
Not onl y i s ther e th e dange r o f generatin g artifact s i n th e proces s o f
preparing th e specime n fo r observation , bu t th e dat a nee d a statistical -
morphometric approac h t o hav e convincin g significance . I n othe r words ,
on ho w man y globule s wa s th e surfac e structur e wel l resolve d an d ove r
what proportio n o f thei r surfaces ? An d o f thes e globules , ho w man y
showed alteratio n i n MLG M an d ove r wha t proportio n o f thei r surfaces ?
Such studie s ar e tediou s an d seldo m done . I n thi s situation , i n whic h i t is
certain that some of the globules are quite old and that even fresh ones may
not reflect thei r condition a t secretion accurately , i t is difficult t o draw firm
conclusions regarding MLGM structural stability. We suggest that a marker
for this purpose migh t be xanthine oxidase . This enzyme i s located largely ,
if no t completely , o n th e inne r surfac e o f th e MLGM , i n th e so-calle d coa t
layer (Freudenstei n et ai, 1979) . To th e exten t tha t i t increases i n th e mil k

serum, disruptio n o f th e membran e i s indicated . B y thi s w e d o no t mea n
enzymic activit y o f xanthin e oxidase , whic h ca n b e misleadin g becaus e i t
is redo x sensitive , bu t rathe r shift s i n distributio n o f actua l mas s o f thi s
protein.
In suppor t o f Wooding* s contention , i t wa s foun d tha t short-interva l
milking o f goat s t o produce fres h mil k resulte d i n a progressive reductio n
in the amoun t o f membran e materia l i n th e ski m mil k (Patto n et a/., 1973) .
Two goat s wer e milke d a t hourl y interval s fo r 1 0 h r afte r a complet e
milking. Concentration s o f phospholipi d an d cholestero l i n th e ski m mil k
were use d a s criteri a o f membran e concentratio n an d thi s wa s supporte d
by the fac t tha t there wa s close correlatio n i n th e change s i n concentratio n
of th e tw o lipids . Durin g th e first five hourl y milkings , th e amoun t o f
membrane i n th e ski m mil k droppe d t o approximatel y 60 % o f th e con -
centration in skim milk of the complete milking . After five hourly milkings ,
the membran e concentration s tende d t o platea u i n on e goa t an d becam e
somewhat errati c i n th e other . Thus , ther e i s th e implicatio n tha t i n th e
udder o f th e goat , MLG M i s t o som e exten t unstabl e an d i s i n par t she d
into th e ski m mil k phase . O f course , mil k fa t globules ma y no t be th e onl y
source o f membran e i n th e ski m mil k phase , an d ther e i s som e evidenc e
that ther e coul d b e specie s differences , a s i s discusse d fo r incidenc e an d
stability o f cytoplasmi c crescent s o n mil k fa t globule s (Sectio n IV ) an d o n
effect o f refrigerate d storag e o n MLG M (Sectio n VII , B) .
B. Changes after Expression from the Gland
1. Cooling
It is reasonable t o expec t tha t changes i n th e temperatur e o f mil k wil l

produce expansion an d contraction o f mil k fa t globules and changes i n th e
MLGM. Fo r reason s o f keepin g quality , th e first procedur e applie d t o
industrial (cow ) mil k i s cooling . Thi s result s i n a phenomeno n whic h
illustrates th e delicac y o f MLG M changes ; namely , th e adsorbtio n ont o
milk fa t globule s o f a substanc e calle d "agglutinin " whic h i s importan t t o
the creamin g phenomenon . Th e deepes t crea m layer s ar e forme d rapidl y
on mil k tha t i s coole d (O-S'^C) . I t becam e possible , b y separatin g mil k a t
different temperatures , to make cream and skim milks that were agglutini n
rich o r agglutini n poor . Whe n recombined , th e agglutini n poo r compo -
nents forme d a crea m laye r ver y slowl y an d inadequately . Th e precis e
nature o f agglutini n ha s no t bee n established , bu t i t appear s t o b e a
member o f th e immunoglobi n M class o f antibodie s (Eube r an d Brunner ,
1984). Fro m a practical standpoint, th e phenomenon o f creaming is mainly
of historical interes t in that the depth o f th e cream layer on a bottle of mil k
(richness) los t it s significanc e whe n homogenization , opaqu e containers ,
and dietar y concer n abou t fa t becam e prevalent . I t i s interestin g tha t
creaming i s greatl y delaye d i n goat' s milk . Whil e a crea m laye r wil l for m
within 1 5 o r 2 0 mi n o n cow' s milk , i t require s hour s o f holdin g fo r goat' s
milk. Thi s i s no t s o muc h du e t o th e smalle r siz e o f goa t globules , whic h
is true, as it is due t o slowness of th e globule s i n clumping together . Fo r thi s
reason, goat milk is said to be naturally homogenized . T h e earlie r literatur e
on creamin g o f cow milk ha s been reviewe d extensivel y b y Brunner (1965) .
No doub t ther e ar e man y temperature-sensitiv e adsorbtion -
desorbtion phenomeno n i n additio n t o agglutini n bindin g transpirin g i n
milk. Anothe r facto r relate d t o thi s i s the progressiv e crystallizatio n o f th e
glycerides i n th e globul e cor e an d o f th e lipid s i n th e MLG M a s mil k cools .
We expec t thi s t o chang e th e positio n an d configuratio n o f som e mem -
brane components : i n som e cases , irreversibly . Moreover , w e expec t a n
accompanying los s o f membran e fluidity i f suc h fluidity exists .
As cow milk is held a t 0-5°C , th e amount o f lipid recovere d i n th e ski m
milk o n centrifuga l separatio n increase s graduall y ove r 4 8 h r (Patto n et a/.,
1980b). Thi s lipi d wa s measure d b y solven t extractio n an d weighing . I t i s
most likel y membran e derive d an d indicate s th e sluffin g o f MLGM . I n thi s
connection, i t i s o f interes t tha t ski m mil k o f lowes t cholestero l conten t
should resul t b y separatin g th e freshes t mil k possible .
2. Agitation, Aeration, and Off-Flavor Development

A natura l accompanyin g facto r i n removin g mil k fro m th e bovin e
gland i s a certain amoun t o f turbulence . T h e splashin g an d foamin g i n th e
glass-walled receivin g vesse l o f a milkin g lin e i s a n obviou s example . I n
addition, whe n th e mil k i s pumpe d fro m th e receive r t o th e refrigerate d
holding tank , ther e wil l b e furthe r agitatio n b y th e pum p an d ofte n ai r
incorporation a s well. These event s se t th e stag e fo r furthe r change s i n th e
MLGM includin g additiona l los s o f membrane , som e denudin g o f th e
globule core , possibl e adsorbtio n o f lipas e fro m th e mil k serum , an d
resulting lipolysi s o f glycerides . T h e incorporatio n o f ai r i s conduciv e t o
oxidation o f th e membran e lipids . These an d othe r factor s influencin g th e
MLGM ar e discusse d below .
The (hydrolytically ) ranci d of f flavor i n whic h MLG M play s a n impor -
tant rol e result s fro m th e actio n o f lipoprotei n lipas e on th e triacylglycerol s
of the globule, with release of butyric and othe r short-chai n acids . From th e
olfactory characte r o f th e of f flavor i t i s clea r tha t butyri c aci d play s th e
dominant rol e an d onl y trac e amount s nee d b e released . Fo r ranci d flavor
to develop , th e triacylglycerol s bearin g butyrat e i n th e lipi d cor e o f th e
globule mus t b e accessible . Sinc e th e cor e normall y i s protecte d b y a laye r
of MLGM , acces s o f th e lipas e i n th e mil k seru m i s denied. Moreover , i t is
not clea r ho w muc h o f a barrier th e nativ e surfac e o f th e fa t droplet , lyin g
under th e MLGM , ma y presen t t o th e lipase . Thi s appear s t o represen t a
monolayer o f pola r lipid s an d protein s (se e Sectio n III) . Bu t thi s surface ,
as wel l a s th e MLGM , ca n b e disrupte d b y agitatio n an d aeration , thu s
allowing th e lipas e t o bin d t o it s substrate . Thes e force s ar e particularl y
pernicious becaus e the y generat e ai r bubble s tha t dissipat e thei r fre e

surface energ y b y strippin g membran e fro m lipi d globules . Th e globul e
surface ca n b e furthe r fracture d b y pump s whic h ca n hav e a n homoge -
nizing actio n unde r th e condition s o f thei r operation . Fo r thi s reason ,
much effor t ha s gon e int o desig n o f pipelin e milkin g system s tha t wil l
minimize agitatio n an d aeratio n o f milk .
Another dimensio n o f th e rancidit y proble m i s variatio n i n suscepti -
bility o f th e mil k amon g individua l cows . Som e mil k taste s ranci d a s i t
comes fro m th e udder . Whethe r thi s i s simpl y a matte r o f "no t enoug h
MLGM t o g o around " is no t known . However , i t i s evident tha t nearl y al l
milk normall y present s enoug h o f a barrie r t o lipas e actio n o n it s fa t
globules tha t rancidit y i s no problem . A n exceptio n t o thi s i s th e effec t o f
warming cold mil k (0-5®C ) t o approximately 3 0 X an d the n recooling . O n
standing, man y individua l mil k sample s s o treate d wil l becom e ranci d
(pertinent literatur e reviewe d b y Brunner , 1965) . Thi s sound s ver y muc h
like a desorbtio n o f surfac e substance s fro m th e globule s i n th e warmin g
and a selective adsorbtio n o f lipas e ont o th e globule s fro m th e mil k seru m
on recooling . Momentar y heatin g t o 6 0X substantiall y inhibit s mil k lipas e
and pasteurizatio n (61.8° C fo r 3 0 mi n o r 71.8° C fo r 1 5 sec ) completel y
inactivates it .
3, Oxidized Flavor
Oxidized flavor result s fro m oxidativ e degradatio n o f polyunsaturate d

fatty acid s containe d i n lipid s o f th e MLGM , particularl y linoleat e an d
arachidonate. A complex , fre e radica l mechanis m involvin g thes e fatt y
acids, oxygen , ascorbi c acid , an d cupri c ion s lead s t o fragmentatio n o f th e
hydrocarbon chains . Ascorbi c aci d i s presen t i n mil k fro m th e udde r a t a
concentration o f 20-30 mg/liter . Mil k is saturated wit h air as a result of th e
milking and pumpin g processes . Ferri c ions and light also can play catalytic
roles i n developmen t o f thi s of f flavor. Resultin g compound s causin g th e
flavor ar e alk-2-enals , particularl y 2-nonenal , 2-octenal , an d 2-heptenal .
Flavor o f an y on e o f thes e i s detectable a t a fe w ppm . I n vie w o f th e hig h
flavor potenc y o f thes e aldehydes , oxidize d flavor ca n b e produce d wit h
very limite d destructio n o f th e lipid s involved . Th e us e o f copper-bearin g
metal alloys in milk plants earlier in this century led to widespread oxidize d
flavor in the milk supply, even to the point that in some communities, it was
not onl y accepted , bu t understoo d t o b e th e norma l flavor o f milk . Th e
introduction o f stainles s stee l int o th e dair y industr y largel y overcam e thi s
problem. However , exposur e o f mil k t o ligh t fo r lon g period s i n super -
market cabinet s ca n induc e thi s of f flavor a t times , an d spontaneou s
outbreaks of th e problem, presumabl y du e t o feed an d metaboli c effects i n
animals, ar e encountered . A mor e detaile d discussio n o f oxidize d flavor i s
provided b y Bading s (1984) .
Because o f a lack of commercia l processin g an d consume r complaints ,
human mil k ha s few flavor problems . A s i s wel l known , huma n mil k fa t
contains no short-chain fatt y acids, so rancid flavor would no t be a problem

in any event. However , it seems likely that food flavors and metabolites may
gain acces s t o huma n mil k just a s easily a s in th e co w becaus e o f th e direc t
monogastric rout e t o th e circulatio n i n th e human . Bu t whethe r th e
consumers objec t t o th e flavor a t time s i s no t known .
4. Freezing
Freezing disrupt s th e structur e o f mil k lipi d globules . Whethe r thi s i s

caused b y expansio n o f th e globul e cor e du e t o crystallizatio n o f th e
triacylglycerols o r t o penetratio n o f th e MLG M b y ic e crystals , o r both , i s
not known . I t i s commonl y observe d tha t whe n froze n mil k (nonhomog -
enized) i s thawed , th e fa t globule s hav e clumped . I f thes e clump s ar e
further warmed , oi l droplet s form . Thus , freezin g mus t creat e break s i n
the membran e s o tha t th e cor e triacylglycerol s ca n emerg e an d coalesc e
with thos e release d fro m othe r globules . Thi s form s th e basi s o f th e
freeze-thaw metho d o f preparin g MLG M (Sectio n VI , A) .
5. Heating
As mil k i s heate d fro m 3 7 t o 50° C change s i n th e MLGM , i f any , ar e

minor. Abov e thi s temperature, inactivatio n o f th e mor e sensitiv e enzyme s
begins. Fo r example , pasteurizatio n (TLS^' C fo r 1 5 sec ) bring s abou t
complete inactivatio n o f mil k lipas e an d alkalin e phosphatase . A majo r
fraction o f thi s latte r enzym e i s i n th e MLGM . A t abou t 72°C , releas e o f
hydrogen sulfid e fro m mil k fat globules commences. The specifi c sourc e o f
this volatile sulfide ha s not been identified bu t xanthine oxidase, a principal
component of the MLGM and one ric h in sulfur, i s a logical candidate. Th e
so-called agglutinin , whic h promote s creaming , i s progressively denature d
as hea t treatmen t proceed s beyon d pasteurization . O n cooling , suc h mil k
slowly form s a shallow, indistinc t crea m layer . Unlik e freezing , heatin g o f
itself, eve n t o boiling , doe s no t disrup t th e physica l integrit y o f mil k lipi d
globules suc h a s to bring abou t significan t oilin g of f o f th e triacylglycerols .
6. Effects of Surface Active Agents
The force s whic h hol d th e MLG M togethe r an d boun d t o th e globul e

core ca n b e overcom e b y variou s surfactant s wit h o r withou t hea t treat -
ment. Eve n low-molecular-weigh t organi c molecule s showin g solubilit y i n
both oi l an d wate r ca n releas e th e membran e (Dappe r et al., 1987) . Tw o
methods fo r preparin g MLG M base d o n detergen t treatment , on e wit h
sodium deoxycholat e (Hayash i an d Smith , 1965 ) and the other with Trito n
X-100 (Patton , 1982) , hav e bee n developed . Th e latte r i s capabl e o f
retaining membrane-bound concanavili n A , a lectin with strong affinity fo r
mannosyl- an d glucosyl-containin g glycoproteins , durin g th e isolation .
Conjugated bil e salts, such as those involve d i n the physiolog y o f digestion ,
will also remove membran e fro m mil k lipid globule s (Patto n et ai, 1986) .
Methods of preparin g MLG M using surfactants ar e discussed unde r Sec-
tion VI, A. Highe r concentrations o f surfactants, especially when coupled
with heat treatment, cause both release of th e membrane and dissociation
of it s components . Th e powerfu l detergent , SDS , i s use d t o completel y
dissociate protein s fro m membran e t o facilitat e thei r electrophoretri c
analysis.
C. Change s Due to Processing
Processing o f mil k an d mil k product s ha s effect s o n th e MLG M du e t o

combined effects of heating, cooling, freezing, and agitation, both with and
without ai r incorporation . Ther e ha s bee n littl e researc h i n thi s are a
dealing specificall y wit h th e membran e becaus e o f th e comple x variable s
and changes involved. Example s of thes e are in the production o f butter,
homogenized milk , an d ic e cream . A brie f summar y o f th e effect s suc h
processing i s understoo d t o hav e o n th e MLGM , togethe r wit h possibl e
functions o f th e membran e i n th e product s involved , follows . Mor e ex -
tensive discussion s o f th e physic s of thes e processe s ar e give n b y Walstra
and Jenness (1984) .
1. Churning
No doubt the production of butter happened by accident thousands of
years ago when some beast of burden carried a container of milk over some
distance. Fundamentally , agitatio n i s all tha t is needed, an d i f i t is main-
tained long enough, butter will be produced. Th e proces s is also aided by
the incorporatio n o f air , hig h fa t content, suc h a s in heav y cream , an d a
suitable temperature. When air bubbles are suspended i n a liquid, such as
milk or cream, the y will tend t o take u p surface activ e material s i n order
to lower their free surface energy and become physically more stable. One
of th e substance s tha t bind s t o th e ai r cell s unde r thes e condition s i s
MLGM. This tends to denude lipi d globule s and expose thei r underlyin g
triacylglycerols. A t the churning temperatur e (approximatel y 12''C ) these
patches o f glyceride s ar e semisoli d (sticky ) an d ten d t o adher e t o on e
another, especiall y unde r th e rigorou s physica l agitatio n o f th e churn .
Eventually th e growin g globul e aggregate s destabiliz e th e air cells (foam )
and, with continued churning, these aggregates become particles of butter.
Much of th e membrane , bu t not all, goes into the buttermilk. Membran e
is stil l retaine d b y som e o f th e fa t globule s i n th e butte r a s see n i n
freeze-fracture replica s in the electron microscope (W. Buchheim, personal
communication). I t i s fel t tha t th e MLG M remainin g i n th e butte r help s
with th e incorporatio n o f moistur e durin g th e workin g proces s an d th e
creation o f a product tha t feel s smoot h o n th e tongue .
People wh o wor k wit h man y huma n an d co w mil k sample s notic e

differences i n tendencie s o f sample s t o chur n spontaneously , eve n whe n
the sample s ar e al l handle d i n th e sam e way . Th e reason s fo r th e differ -
ences ar e no t known . Relativ e softnes s o f th e fa t a t a given temperatur e i s
known t o b e on e facto r i n churning . Thus , liqui d t o soli d rati o o f mil k
triacylglycerols ma y b e a facto r i n th e huma n i n whic h die t varie s greatly ,
but difference s ca n b e perceive d i n th e tendenc y t o chur n amon g mil k
samples o f cow s receivin g th e sam e ration . Thus , mor e subtl e individua l
differences mus t als o b e involved .
2. Homogenization
The discover y tha t homogenizatio n stabilize d th e suspensio n o f fa t i n
milk, mad e th e mil k tast e creamier, increase d milk' s resistance t o oxidize d
flavor, an d lowere d th e cur d tensio n mad e fo r rapi d adoptio n o f th e
process. Homogenizatio n i s effecte d b y pumpin g mil k a t hig h pressur e
through narro w orifice s a t temperatures approximatin g thos e fo r pasteur -
ization (65-80°C) . Thi s produce s drasti c turbulen t an d cavitationa l force s
which physicall y disintegrat e th e globules , particularl y th e large r ones ,
leading t o a reduction i n thei r mea n diamete r fro m 3 or 4 [A m to less tha n
1 [im . Th e expose d ne w triacylglycero l surface s adsor b mil k proteins ,
particularly casei n micelles . Abou t 30 % of th e casein i n homogenize d mil k
is associate d wit h th e lipi d globules . Th e resultin g globul e suspensio n i s
probably stabilized in two ways. The adsorbe d protei n increases the densit y
of th e globules , thu s overcomin g thei r tendenc y t o rise , an d th e negativ e
charge o f adsorbe d casei n micelle s probabl y make s th e globule s mor e
self-repellent. I t is also possible that such clumping factor s as agglutinin ar e
denatured i n th e homogenizing . I n an y event, th e norma l tendenc y o f th e
globules t o clump seem s t o be destroyed . However , a study (Keena n et ai,
1983) has made it clear that a large proportion o f globule s in homogenize d
milk stil l retai n membran e material , bot h protei n an d lipid , an d tha t uni t
(normal biological ) membran e structur e ca n b e observe d o n the m i n th e
electron microscope .
3. Ice Cream Manufacture
From formulation , processing , an d physicochemica l standpoints , ic e

cream i s on e o f th e mos t comple x foods . Whil e detaile d consideratio n o f
the produc t i s beyond th e scop e o f thi s article, we should not e tha t MLGM
plays a role i n its processing an d properties . Afte r blendin g of ingredients ,
heating, homogenizing , an d coolin g th e basi c ic e crea m mix , i t i s froze n
while bein g whippe d t o incorporat e air . Thi s produce s a syste m o f suga r
syrup containin g fa t globules , proteins , ic e crystals , an d ai r cells . I t i s
important tha t th e ai r cell s b e smal l an d stable . A s a n emulsifyin g agent ,
MLGM i s considere d t o b e a n importan t componen t i n achievin g thi s
condition. Sweet cream buttermilk, which is rich in MLGM and sometimes

used a s a sourc e o f mil k solid s i n ic e cream , i s sai d t o impar t excellen t
whipping propertie s t o th e mix . So-calle d emulsifying-whippin g agent s
are often adde d t o the ic e cream mix . They includ e mono - and diglycer-
ides, Tweens and Spans, and lecithin (phosphohpid)-containin g products .
These hav e propertie s lik e thos e o f th e pola r lipid s i n MLGM . T o ou r
knowledge, no one has evaluated the specific contribution of MLGM to the
body and texture of ic e cream. However , i n this era of decreasing th e fat
content, with the hope that palatability can be maintained, it seems worthy
of investigation .
Acknowledgements
Research o f S. P. is supported b y funds fro m th e Nationa l Dair y Promotion an d Researc h

Board. Researc h o f T . W . K . i s supported b y Gran t GM3124 4 fro m th e Nationa l Institut e o f
General Medica l Science , N I H , an d b y a gran t fro m th e Nationa l Dair y Promotio n an d
Research Board .
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B. Particulat e Constituent s
in Huma n an d Bovin e Milk s
ROBERT G. JENSEN
BERNARD BLANC
STUART PATTO N
I. Introductio n
Milks ar e biological fluids o f exceptiona l complexit y containin g thousand s

of compounds . Thes e ar e locate d i n severa l compartments , directe d ther e
by th e biologica l an d physicochemica l force s actin g durin g mil k synthesis ,
secretion, an d thereafter . Compartmentatio n i s on e o f th e factor s whic h
control th e flow o f mil k component s an d thei r product s int o an d throug h
the digestiv e an d absorptiv e system s o f th e consumer . Th e compound s i n
milk provid e nutriture , structura l component s fo r cellula r membranes ,
and nonnutritiv e messages , e.g. , immunologica l system s fo r hos t defense .
The compartments i n bovine milk , which is produced an d consumed i n th e
largest quantity , ar e altere d b y processing . Mos t o f th e mil k i s processed ,
i.e., pumped , agitated , pooled , cooled , clarifie d (centrifugatio n t o remov e
cells, etc) , th e fa t conten t standardized , usuall y t o a lowe r amoun t b y
controlled separatio n (centrifugation) , pasteurized , an d homogenize d t o
reduce th e siz e o f th e fa t globules . Unfortunately , compartmentatio n i n
H A N D B O O K O F MIL K COMPOSITIO N
Copyright ® 199 5 b y Academi c Press , inc.
All righ u reserved . N o reproductio n withou t permission .

The Structure of Milk: Implications For Sampling and Storage

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CHAPTER 2

The Structure of Milk:

I. Intracellula r Origi n an d Growth o f Milk Lipi d

Membrane an d membrane-associate d materia l whic h surround s th e

Earliest intracellula r precursor s o f mil k lipi d globule s appea r t o originat e

By whateve r mechanis m the y originate , mil k lipi d globul e precursor s first

identical polypeptid e patterns . Man y polypeptide s wit h electrophoreti c

II. Rol e of Intracellular Lipi d Drople t Coa t

Coat materia l o n surface s o f intracellula r lipi d droplet s undoubtedl y i s

droplet-cytoplasmic lipi d drople t fusion s ar e commonl y see n i n electro n

III. Mil k Lipi d Globule Secretio n

The proces s o f lipi d drople t secretio n ha s bee n describe d repeatedl y sinc e

resulted i n formatio n o f intracellula r vacuole s containin g membrane -

IV. Natur e an d Frequency of Cytoplasmi c

A smal l compartmen t o f mil k tha t ha s receive d limite d attentio n i s cres -

A. Mode of Crescent Formation

In th e proces s o f thei r secretion , mil k fa t globule s usuall y ar e envelope d

Figure I Electro n micrograp h o f a typica l huma n mil k fa t globul e bearin g a crescen t o f

diurnal variatio n i n th e human , wit h greates t number s o f crescent s bein g

B. Properties of Cytoplasmic Crescents

Cytoplasmic crescent s hav e bee n observe d t o contai n al l th e variou s mem -

milk removal , deterioratio n o f crescent s ca n procee d i n th e gland . Th e

C. Detectio n and Quantification of Crescents

Jannsen an d Walstr a (1982 ) originated us e of the fluorescent dye , acridine

agents; a t highe r magnifications , th e globule s nee d t o b e concentrate d

It ma y see m unlikel y tha t somethin g a s quantitativel y limite d a s crescent s

important molecule s ar e effectiv e a t ver y lo w level s includin g enzymes ,

V. Siz e and Membrane Are a Distributio n of Mil k

these globule s mus t accoun t fo r mos t o f th e secretor y events , the y contai n

B. Membrane Surface Area

globule membran e (i.e. , one sid e o f tha t membrane) . However , ther e is , as

V I . Natur e o f th e Mil k Lipi d Globul e Membran e

A. Isolation of Milk Lipid Globule Membrane

The membran e surroundin g lipi d globule s i n mil k closel y resemble s

Figure 2 Electro n micrograp h o f a co w MLG M preparation . Thi s preparatio n consist s

collected b y centrifugation a t g force s u p t o or exceedin g 100,000 . Alter -

1983). Mil k lipi d globule s an d membrane s derive d therefro m ar e identica l

Over 95 % of th e tota l lipid s i n mil k fro m cow s (Huan g an d Kuksis , 1967 ;

material at temperatures below the solidification poin t of the triacylglycero l

Constituent clas s Unit Cow'' Human*

''Taken fro m compilatio n i n Keena n et al. (1988) .

surface membranes , isolated fro m homogenate s o f mammar y glan d as well

Constituent clas s CoW Human *

''As compiled i n Keena n et al. (1988); Patton and Keena n (1975).

Phospholipids o f mil k ar e mainl y presen t i n lipi d globule s (abou t 60%

(about 40% ) (Huan g an d Kuksis , 1967 ; Patto n an d Keenan , 1971) . Th e

functional role s i n a numbe r o f biologica l phenomena , suc h a s cell-cel l

60-390 day s postpartum , thi s rati o wa s greate r tha n 3 . Mil k collecte d a t

4, Fat-Soluble Vitamins and the Membrane

Certain o f th e vitamins . A, D , E, and K are sai d t o be fa t soluble . Whe n

Consideration i s given her e mainl y t o protein s o f th e huma n an d co w mil k

borne i n min d tha t th e majo r huma n an d co w globul e proteins , suc h a s

1. Compartmentation of Globule Proteins

2. The Principal Globule Proteins

Figure 3 A n SD S ge l comparin g protein s o f huma n (H ) an d bovin e (B ) mil k lipi d globules .

and butyrophilin i n both the human an d th e cow samples. This is generally

a. Mucin(s) . Hig h molecula r weigh t glycoproteins , no w know n a s ep -

and Keena n (1975 ) fo r th e co w MLG M proteins . I t appear s t o b e o f th e

b. Xanthin e oxidase (monomer , 155,000 ; band I of Figur e 3) . O n gels , thi s

and carbohydrat e compositio n (Ima m et al, 1981) . I n th e nativ e state ,

c. Butyrophili n (ban d 3 o f Figur e 3) . Thi s protein , with M^ of 66,000 ,

d. M r 62,500 (band 4 of Figure 3) . Thi s protei n ma y be a close structura l

e. M, 46.00 0 t o 52,00 0 (ban d 5 o f Figur e 3 ; als o know n a s PAS-V I an d