original communications

242 The American Journal of Clinical Nutrition 29: MARCH 1976, pp. 242-254. Printed in U.S.A.
Copper, iron, and zinc contents
of mature human milk3
Mary Frances Picciano,4 Ph.D., and Helen A. Guthrie,5 Ph.D.
ABSTRACT Daily, weekly, and within-day variations in copper, iron, and zinc contents of
human milk were investigated in order to determine whether one sample from an individual is
representative of these elements. Total solids, fat, and protein contents were also measured. Fifty
women in their 6th to 12th week of lactation each provided seven milk samples consisting of five
consecutive daily samples and two additional samples collected either within a single day or at
weekly intervals. Fat varied the most of all constituents and total milk solids reflected this
variability. Values ranged from 0.2 to 10.4 g/ 100 ml for fat and from 8.58 to 17.49 g/ 100 ml for total
solids. Protein varied from 0.76 to 2.04 g/l00 ml among individuals, with little variation within an
individual. Copper content varied considerably among women and within the same woman. With a
large proportion of low values, the range was 0.09 to 0.63 g/ml. Iron content was also found to
vary within women as well as among women. Values ranged from <0.1 to 1.6 zg/ml with a
preponderance of low values. Zinc content was more evenly distributed over the range of 0.14 to 3.95
.eg/ml, and within an individual it did not vary widely. A representative estimate of copper and
iron contents would therefore require multiple samples, whereas only one sample may provide a
representative estimate of zinc content. Comparison of morning, midday, and evening values showed
that copper and zinc are higher in the morning and iron is lower at this time. Increased amounts of
copper, iron, and zinc were found in multiparous women whether or not they had previously
lactated. Milk from older women had lower iron and higher copper and zinc contents than that from
younger women. No differences were found in milk of women receiving dietary mineral and vitamin
supplements. Calculations indicated that fully breast fed infants under 3 months of age receive
approximately 0.35 mg/kg per day of zinc and 0.05 mg/kg per day of both copper and iron. Am.
J. C/in. Nutr. 29: 242-254, 1976.
The composition of human milk is of
interest to those concerned with the nutrition
of infants, since early feeding regimens are
based on its use or substitutes modified to
resemble it. However, it has long been known
that human milk composition varies widely
among women and that variations in quality
even occur within the same woman. If human
milk is to be used as a standard, it is
important to establish which nutrients vary,
the extent of the variations, possible factors
exerting an influence, and the effects such
variations may have on the growth of the
infant. Early investigators (1-6) have identi-
fied variations in the macronutrients of
human milk as well as some factors which
influence their composition. Considerably
1 From the Nutrition Program, Division of Biological
Health, Pennsylvania State University. Taken in part
from a thesis submitted by M. F. Picciano to the
Graduate School, Pennsylvania State University, in
partial fulfillment of the requirements for the Ph.D.
degree.
2 Address reprint requests to M. F. Picciano, 457
Bevier Hall, School of Human Resources and Family
Studies, University of Illinois, Urbana, Illinois 61801.
Authorized for publication on May 5, 1975 as paper
no. 4860 in the journal series of the Pennsylvania
Agricultural Experiment Station.
Assistant Professor of Nutrition, University of Il-
linois. 6 Professor of Nutrition, Pennsylvania State
U niversity.

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CU, FE, AND ZN I N MATURE HUMAN MI L K
243
less inform ation is available for the m icronu-
trients, especially the trace elem ents. That
voids exist in our know ledge of trace elem ent
com position of hum an m ilk has recently been
stressed by the W orld H ealth O rganization
E xpert C om m ittee on Trace E lem ents in
H um an N utrition (7). The C om m ittee recom -
m ended that highest priority be given to the
analysis of hum an m ilk in an international
collaborative effort to obtain data on the
trace elem ent content of foods.
The purpose of this investigation w as to
determ ine the copper, iron, and zinc contents
of hum an m ilk and to identify sources of
variation, since reported values vary w idely.
The range of reported m ean values (zg/m l)
for copper is 0.22 to 1.5 (8-11); for iron, 0.44
to 5.0 (9-13); and for zinc, 1.3 to 12.4
(10-12). From an analysis of 22 sam ples, the
m ost recent investigation (10) provided aver-
age values for copper, iron, and zinc of 0.24,
0.84, and 1.34 zg/m l, respectively. W hether
these reported variations could be attributed
to individual differences am ong w om en or
differences w ithin the sam e w om an w ere
exam ined. W ithin individuals, patterns of
variation throughout a day, from day to
day, and from w eek to w eek w ere studied.
From such inform ation, it is possible to
determ ine w hether one sam ple of m ilk from a
particular individual m ay be considered rep-
resentative of copper, iron, and zinc contents.
In addition, influences of m aternal age, pari-
ty, and lactation history w ere exam ined.
Subj ect s and methods
Subjects
Fifty P ennsylvania m others in the 6th to 12th w eek of
lactation each provided seven m ilk sam ples for analyses.
A ll subjects had healthy full-term infants and w ere
feeding them exclusively on breast m ilk w ithout any
supplem entary food. Forty-tw o subjects w ere betw een
the ages of 20 and 30, while eight were over 30. Sixteen
subjects were primiparae and 34 were multiparae, of
w hom four w ere lactating for the first tim e.
Maternal nutrient intake
To obtain an indication of the nutritional quality of
subjects diets, 24-hr recalls w ere obtained at the tim e of
interview . N utrient intakes w ere tabulated using data
cards from the U .S . D epartm ent of A griculture based on
their Home and Garden Bulletin 72 (14). A dditional
cards w ere prepared as needed from data in Agricultural
Handbook 8 (15) and Food Values of Portions Com-
monly Used (l6). A nalyses for kilocalories, protein,
vitamin A, thiamin, riboflavin, ascorbic acid, calcium,
and iron w ere m ade. S ince com plete data are not
available for copper and zinc contents of foods, these
nutrients w ere not tabulated. C om parison w ith R ecom -
m ended D ietary A llow ances (R D A ) (17) show ed that
w ith the exception of kilocalories and iron, average
intake either m et or exceeded the R D A . H ow ever,
average intakes did provide tw o-thirds of the R D A for all
nutrients tabulated, a level of intake judged to be
adequate.
Thirty-seven subjects (74% ) w ere also supplem enting
their diets w ith vitam in-m ineral preparations. A ll of
t h e s e c o n t a i n e d 3 0 mg o f i r o n , a n d f i v e wo me n ( 1 0 %)
w ere taking tw o such preparations. A few supplem ents
contained m inim al am ounts of copper and less than 10%
of the R D A for zinc.
Collection of samples and experimental design
S am ple collections w ere m ade betw een A pril and
A ugust of 1972. A s m em bers of nursing m other groups,
subjects were instructed to use both breasts at a feeding.
The breast used first at any one feeding w as em ptied and
used second at the follow ing feeding. W hile m others
adhered to this practice, all sam ples w ere collected w ith a
polyethylene hand breast pum p or by m anual expression
from the second breast before it w as offered to the infant.
In order to m inim ize the possibility of contam ination,
sam ple collection equipm ent w as acid-w ashed, rinsed in
glass-redistilled w ater, and enclosed in plastic. S ubjects
also received explicit verbal and w ritten instructions
concerning necessary precautions w hile collecting sam -
ples. S am ples w ere frozen im m ediately after collection
and kept frozen until analyzed.
S ubjects each provided one 40-m I sam ple of m ilk a
day, at the early m orning feeding, for 5 consecutive days
(daily period). Tw o additional sam ples w ere provided
either at w eekly intervals, also collected at the early
m orning feeding (w eekly period), or w ithin a single day,
collected at m idday and evening feedings (w ithin-day
period). All 50 participants therefore each supplied seven
sam ples of hum an m ilk according to the experim ental
design outlined in Table I. Early morning samples were
collected betw een the hours of 5:00 and 8:00 AM wi t h a
4- to 8-hr lapse from previous feedings. M idday sam ples
w ere collected around the noon hour and evening sam ples
at approximately 6:00 PM. Since all subjects practiced
the dem and m ethod of nursing, there w as only a 2- to
3-hr lapse from previous feeding in the w ithin-day period.
A nalttical methods
D uplicate 5-m I portions of w hole m ilk w ere placed in
a beaker which had been dried to constant weight. The
milk was then dried over a steam bath and in an oven at
97 to 100 C for 3 to 4 hr. A fter the sam ples had been
cooled in a desiccator, total m ilk solids w ere determ ined
gravimetrically (18). S ubsequently, the sam e sam ple w as
digested w ith concentrated nitric and sulfuric acids in a
4:1 ratio (v/v). Additional nitric acid was added until the
digest was clear. The clear sam ple w as brought to a
volume of 5 ml with glass-redistilled water and directly
aspirated into a P erk in-E lm er m odel 403 atom ic absorp-
tion spectrophotom eter for determ inations of copper,
iron, and zinc contents. The spectrophotometric method
of Nakai and Li (19) was used to measure fat and protein
contents.

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244 PICCIANO AND GUTHRIE
TABLE I
Experimental design
Ti me o f c o l l e c t i o n
Ea r l y mo r n i n g Mi d d a y Ev e n i n g
W eek I
Day I
Day II
Within-day Ill S Day 1110
r - I I
ii 1 (____.Day lV0
.SH
8.. W ithin-day IV
I
8.1
. ;I
. I

Day I Weck II
Week III
Dayl
Within-day III
I
Within-day Ill
I
Wi t h i n - d a y I V
I
W ithin-day period (n =
Wi t h i n - d a y I V
10)
Wi t h i n - d a y p e r i o d ( n = 1 5 )
W ithin-day samples were collected on either day III or day IV of t h e d a i l y p e r i o d .
S tatistical m ethods
The total variance of each milk constituent was
quantitatively partitioned among the sources of variation
and expressed as percentage of the total. Sources of
v a r i a t i o n we r e a mo n g mo t h e r s , a mo n g s a mp l e s wi t h i n
mothers, and among duplicates within samples (experi-
mental error). An analysis of variance with a random
effects model and a hierarchical classification of data was
used to measure the relative intensities of sources of
variation. Differences among means in the within-day
period were determined by Du n c a n s mo d i f i e d l e a s t
square difference test, and i n f l u e n c e s o f d i e t a r y s u p p l e -
ments, maternal age, parity, and lactation history were
assessed with Students t test (20).
R esults
Analyses of variance for total solids, fat,
and protein contents are presented in Table 2
for daily, weekly, and within-day periods.
Total solids and fat content varied similarly
from day to day and week to week. In each
case, variations among subjects accounted for
50 to 60% and variations among samples
accounted for 40 to 50%. In contrast, approx-
imately 80% of total within-day variances for
these constituents is attributable to variations
among samples. In all experimental periods,
only 5% of total variances in protein content
is observed among samples, while among-
subject differences accounted for 93%.
Analyses of variance for copper content
expressed as micrograms per milliliter of
milk, micrograms per gram of milk solids,
and micrograms per gram of solids-not-fat
are presented in Table 3 for daily, weekly,
and within-day periods. All ratios of parti-
tioned variances are significant, yet variation
patterns differed in each experimental period.
Regardless of expression basis, copper con-
tent in the daily period varied in a similar
fashion, with approximately 70% of total
variance occurring among subjects and 22%
occurring among samples within subjects. In
the weekly period, variation in copper
among samples was about 28% and variation
among rubjects was 55% when expressed as
micrograms per milliliter of milk or micro-
grams per gram of solids-not-fat. However,
when considered on the basis of total milk
solids, weekly variance among samples only
accounted for 18% of total and among sub-
jects for 67%. In contrast, within-day copper
variance was greatest among samples when
expressed as micrograms per gram of milk
solids. Approximately 21 % is ascribable to
variations among samples within subjects and
77% to variations among subjects. Micro-
grams of copper per milliliter of milk or per
gram of solids-not-fat varied similarly, with
variations among subjects accounting for
85%, and with variations among samples ac-
counting for only 12%.
Under our conditions of analysis previously
described, the smallest detectable amount of

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TABL E
M eans and analysis of variances for total solids, fat, and protein contents of hum an m ilk
M ilk
constituent
(g/lO O m l)
M ean S D R ange S ource of variance df
M ean
square
F
va1ue
V ariance
% of
O bserved total
D aily period
Total solids
Fat
P rotein
W eekly period
Total solids
Fat
Pr o t e i n
1 1 . 8 5 1.43 8.58- 17.49 Total 499 2.03 100.00
A m ongsubjects 49 13.63 8.500 1.20 59.16
A m ong sam ples 200 1.60 29.390 0.77 38.06
A m ong replicates 250 0.06 0.06 2.78
3 . 0 5 1 . 9 1 0.20-10.00 Total
A m ong subjects
A m ong sam ples
A m ong replicates
499
49
200
2 5 0
23.48
3.22
0 . 0 2
7.300
145.660
3.65
2.03
1.60
0 . 0 2
100.00
55.57
4 3 . 8 2
0 . 6 1
1.28 0.24 0.76-2.04 Total
A m ong subjects
A m ong sam ples
A m ong replicates
499
49
200
2 5 0
0.552
0.007
0 . 0 0 9
76.670
7.680
0.059
0.054
0.003
0 . 0 0 1
100.00
93.05
5.35
1.60
11.70 1.35 8.80-14.67 Total
A m ong subjects
A m ongsam ples
A m ong replicates
149
24
50
75
7.78
1.55
0.02
5 0 3 0
l0l.8I#{176}
1.82
1.04
0.77
0.02
100.00
5 7 . 1 1
42.05
0.83
2 . 9 1 1.68 0.20 7.20 Total
A m ong subjects
Amo n g s a mp l e s
Amo n g r e p l i c a t e s
149
2 4
5 0
7 5
1 1 . 3 4
2 . 8 0
0 . 0 1
4 . 0 6 0
2 0 5 . 6 2 0
2.83
1 . 4 2
1 . 3 9
0 . 0 1
100.00
5 0 . 3 4
4 9 . 1 9
0 . 4 8
1 . 2 5 0 . 2 8 0.79-2.04 To t a l
A m ong subjects
A m ong sam ples
Amo n g r e p l i c a t e s
1 4 9
24
5 0
7 5
0.429
0 . 0 1 0
0 . 0 0 1
43.150
9 . 9 8 0
0 . 0 7 5 4
0.0699
0.0045
0 . 0 0 1 0
100.00
92.74
5.94
1 . 3 2
8 . 5 8 - 1 7 . 4 9 To t a l
A m ongsubjects
A m ong sam ples
A m ong replicates
1 4 9
2 4
50
75
4 . 4 0
2.59
0.02
1 . 7 0
11.650
1 . 6 1
0 . 3 0
1.29
0.02
100.00
1 8 . 7 1
79.90
1.38
3.72 1.90
0.20-10.00 Total
A m ong subjects
A m ong sam ples
A m ong replicates
149
24
50
75
9.983
5.798
0.003
1.72
1840.690
3.598
0.698
2.897
0.003
100.00
19.39
80.53
0.09
1.30 0.22 0.76-2.04 Total
A m ong subjects
A m ong sam ples
A m ong replicates
149
24
50
75
0.2658
0.0049
0.0011
53370
4540
0.0465
0.0435
0.0039
0.0011
100.00
93.47
4.18
2.36
Wi t h i n - d a y p e r i o d
Total solids 12.34 1.27
Fat
P rotein
P < 0 . 0 0 1 .
C U , FE , A N D ZN IN M A TU R E H U M A N M ILK
2 4 5
iron w as 0.1 zg/m l, and 40 sam ples w ere
found to contain less than this am ount. This
does not necessarily im ply that no iron w as
present, and could be explained by insensitiv-
ity of the analytical technique. W hen no
detectable am ounts w ere found, the value
0.05 zg iron/m l of m ilk w as assigned for
purposes of com putation. D ata show ing
m eans and analyses of varia.pce for iron
content of daily, w eekly, and w ithin-day
periods are presented in Table 4. E ven though
variations in each experim ental period are
patterned differently, all ratios of partitioned
variance are significant. M ost of the variation
in iron content during the daily period is
ascribable to variations am ong sam ples
w ithin subjects (53% ) regardless of expression
basis. A m ong-subject variations account for
approxim ately 39% . In the w eekly period,
variances am ong sam ples ranged from only
9% w hen iron w as expressed as m icrogram s
per gram of solids-not-fat to 21 % w hen

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TABL E 3
Me a n s a n a l y s i s o f v a r i a n c e s f o r i r o n c o n t e n t o f h u ma n mi l k s a mp l e s
V ariance
M ean
M ilk constituent M ean SD So u r c e o f variances df uare
F
value0
O bserved of
total
D aily period
Cu ( p g / mI )
C u (pg/g m ilk solids)
Cu ( p g / g s o l i d s - n o t - f a t )
We e k l y p e r i o d
C u ( p g / ml )
C u ( j . e g / g mi l k s o l i d s )
C u ( p g / g s o l i d s - n o t - f a t )
W ithin-day period
C u ( p g / mI )
C u (pg/g m ilk solids)
C u ( p g / g s o l i d s - n o t - f a t )
0.245 0.077 Total 499 0.0060 100.00
Amo n g s u b j e c t s 4 9 0.0464 15.99 0.0045 72.19
Amo n g s a mp l e s 200 0.0029 6.44 0.00 12 20.34
A m ong replicates 250 0.0005 0.0005 7.47
2.08 0.64
2.81 0.92
To t a l
Amo n g s u b j e c t s
Amo n g s a mp l e s
Amo n g r e p l i c a t e s
Total
A m ong subjects
Amo n g s a mp l e s
A m ong replicates
4 9 9
4 9
200
2 5 0
499
49
200
250
3 . 0 6
0.21
0.04
6.08
0.50
0.08
14.34
6.02
12.29
6.01
0 . 4 1
0.28
0.09
0.04
0.85
0.56
0.21
0.08
100.00
69.59
2 1 . 7 5
8.66
100.00
65.94
24.34
9.71
0 . 2 3 2 0.069
1.98 0.56
2.66 0 . 8 1
Total
A m ong subjects
A m ongsam ples
A m ong replicates
Total
A m ong subjects
Amo n g s a mp l e s
A m ong replicates
To t a l
Amo n g s u b j e c t s
A m ong sam ples
A m ong replicates
149
24
50
75
149
24
5 0
75
1 4 9
2 4
50
75
0.0201
0 . 0 0 3 2
0.0006
1.40
0 . 1 6
0.05
2 . 5 5
0.53
0.13
62.34
5 . 1 5
8.85
3.37
4 . 8 6
3.94
0.0047
0 . 0 0 2 8
0 . 0 0 1 3
0.0006
0.31
0.21
0.06
0.05
0 . 6 6
0 . 3 4
0.20
0.13
100.00
59.57
27.66
12.77
100.00
66.87
17.98
15.15
100.00
5 0 . 6 3
29.36
20.01
0.228 0.077
1.85 0.62
2.72 0.97
Total
A m ong subjects
A m ong sam ples
A m ong replicates
Total
A m ong subjects
A m ong sam ples
A m ong replicates
Total
A m ong subjects
A m ongsam ples
A m ong replicates
149
24
50
75
149
24
50
75
149
24
50
75
0.0323
0.00 14
0.0001
1.947
0.170
0.009
5.01
0.28
0.02
23.29
9.64
11.43
18.78
18.12
1.2.76
0 . 0 0 5 9
0 . 0 0 5 2
0.0006
0.0001
0.386
0.296
0.081
0.009
0.938
0.789
0.127
0.022
100.00
87.07
10.50
2.43
100.00
76.74
20.90
2.35
100.00
84.11
13.58
2.31
0 <0 . 0 0 1 .
246 PI CCI ANO AND GUTHRI E
expressed per gram of m ilk solids. V ariations
am ong subjects ranged from 59% (m icro-
gram s per gram of m ilk solids) to as high as
78% (m icrogram s per gram of solids-not-fat).
Thirty-nine percent of the variation in iron
content is attributable to am ong-sam ple vari-
ances and 50% is attributable to am ong-sub-
ject variances in the w ithin-day period.
M eans and analyses of variance for zinc
content are presented in Table 5. A s w ith
copper and iron, all ratios of partitioned
variances for zinc content are significant.
C om parable patterns of variation w ere ob-
served in daily and w eekly periods. M ost of
the total variance (85%) is derived from
am ong subjects, w hile only 13% is derived
from am ong sam ples. D uring the w ithin-day
period, zinc expressed per m illiliter of m ilk or
per gram of solids-not-fat displayed parti-
tioned variances sim ilar to daily and w eekly
periods. H ow ever, nearly tw ice as m uch vari-
ation (24% ) w as observed am ong sam ples

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TABLE 4
Means and analysis of variances for iron content of human milk samples
Variance
Mean
Milk constituent Mean SD Source of variances df square
F
Value#{176}
Observed % of
total
Daily period
Fe (pg/mI)
Fe (pg/g milk solids)
Fe (pg/g solids-not-fat)
W eekly period
Fe (pg/mI)
Fe (pg/g milk solids)
Fe (pg/g solids-not-fat)
W ithin-day period
Fe (pg/mI)
Fe (pg/g milk solids)
Fe (pg/g solids-not-fat)
0.202 0.17 Total 499 0.030 100.00
Among subjects 49 0.158 4.73 0.013 41.32
Among samples 200 0.034 16.73 0.0 16 52.06
Among replicates 250 0.002 0.002 6.62
1.75 1.38 Total
Amongsubjects
Amongsamples
Among replicates
499
49
200
250
9.28
2.27
0.14
4.10
16.29
1.90
0.70
1.06
0.14
100.00
36.85
55.85
7.31
2.35 2.03 Total
Amongsubjects
Amongsamples
Among replicates
499
49
200
250
21.31
4.64
0.27
4.59
17.37
4.12
1.67
2.19
0.27
100.00
40.44
53.07
6.49
0.232 0.190 Total
Amongsubjects
Among samples
Among replicates
149
24
50
75
0.166
0.0 17
0.005
9.64
3.20
0.036
0.025
0.006
0.005
100.00
68.68
16.41
14.91
2.02 1.46 Total
Among subjects
Amongsamples
Among replicates
149
24
50
75
8.81
1.26
0.38
6.99
3.29
2.12
1.26
0.44
0.38
100.00
59.25
20.64
18.04
2.76 2.50 Total
Among subjects
Among samples
Among replicates
149
24
50
75
31.41
1.94
0.79
16.18
2.47
6.28
4.91
0.58
0.79
100.00
78.26
9.21
12.54
0.19 0.35 Total
Among subjects
Among samples
Among replicates
149
24
50
75
0.048
0.0 10
0.001
4.70
8.44
0.012
0.006
0.005
0.001
100.00
52.44
37.48
10.08
1.54 0.75 Total
Among subjects
Among samples
Among replicates
149
24
50
75
2.30
0.42
0.08
5.46
5.46
0.056
0.031
0.0 17
0.008
100.00
55.66
30.60
13.73
2.25 1.31 Total
Among subjects
Amongsamples
Among replicates
149
24
50
75
6.14
1.80
0.17
3.40
10.54
1.716
0.725
0.819
0.172
100.00
42.25
47.74
10.01
CU, FE, AND ZN IN MATURE HUMAN MILK 247
0P <0.001.
when zinc was expressed per gram of milk
solids, with less variability attributable to
among subjects (75%).
Frequency distributions (Figs. 1 to 3) of
copper, iron, and zinc contents were similar in
all experimental periods regardless of expres-
sion basis. Therefore, data were pooled and
presented only on the basis of volume of milk
(milliliters). Since a preponderance of lower
values for copper and iron content were
found, maximum frequencies of 0.20
.tg/ml and 0.10 .tg/ml are slightly lower
than mean values of 0.24 g/ml and 0.21
ig/ml, respectively. Zinc content was more
evenly distributed over the reported range,
with the maximum frequency not different
from the mean value of 1.62 zg/ml.
Mean values for milk constituents of morn-
ing, midday, and evening samples are shown
in Table 6. Lower total solids content was
found during the morning than during either
midday or evening feedings. Fat content

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TABLE 5
Means and analysis of variances for zinc content of human milk samples
Variance
Mean
Milk constituent Mean SD Source of variances df square
F
value0
Ob served % of
total
Daily period
Zn (pg/mI)
Zn (pg/g milk solids)
Zn (pg/g solids-not-fat)
W eekly period
Zn (pg/mI)
Zn (pg/g milk solids)
Zn (pg/g solids-not-fat)
Within-day period
Zn (pg/mi)
Zn (pg/g milk solids)
Zn (pg/g solids-not-fat)
1.68 0.78 Total 499 0.602 100.00
Amongsubjects 49 5.399 35.92 0.525 87.19
Amongsamples 200 0.150 40.75 0.073 12.18
Among replicates 250 0.004 0.004 0.61
14.15 6.26 Total
Among subjects
Among samples
Among replicates
499
49
200
250
342.93
11.69
0.34
29.35
34.60
39.14
33.12
5.67
0.34
100.00
84.64
14.50
0.86
19.25 9.37 Total
Among subjects
Amongsamples
Among replicates
499
49
200
250
769.45
26.14
0.98
29.43
26.79
87.88
74.33
12.58
0.98
100.00
84.58
14.31
1.11
1.59 0.84
13.47 6.69
18.27 9.65
Total
Among subjects
Among samples
Among replicates
Total
Among subjects
Amongsamples
Among replicates
Total
Among subjects
Among samples
Among replicates
149
24
50
75
149
24
50
75
149
24
50
75
3.786
0.189
0.009
239.85
13.31
0.66
500.17
26.57
1.69
20.07
20.55
18.02
20.21
18.83
15.74
0.699
0.600
0.090
0.009
44.74
37.76
6.33
0.66
93.07
78.93
12.44
1.69
100.00
85.83
12.85
1.32
100.00
84.39
14.14
1.47
100.00
84.81
13.37
1.81
1.58 0.81 Total
Among subjects
Among samples
Among replicates
149
24
50
75
3.575
0.174
0.004
20.56
40.45
0.656
0.567
0.085
0.004
100.00
86.42
12.93
0.66
12.81 6.37 Total
Among subjects
Among samples
Among replicates
149
24
50
75
202.56
20.05
0.37
9.85
54.03
40.63
30.42
9.84
0.37
100.00
74.87
24.22
0.91
18.82 10.02 Total
Among subjects
Among samples
Among replicates
149
24
50
75
535.04
31.85
1.06
16.80
30.13
100.32
83.87
15.39
1.06
100.00
83.60
15.35
1.05
0 <0.001.
248 PICCIANO AND GUTHRIE
varied significantly at each feeding, with the
highest value observed in the evening and the
lowest in the morning. Protein concentrations
were lower in morning than in evening milk.
Morning milk was higher in copper and zinc
and lower in iron compared to other feedings
during the day. These differences were not
apparent when minerals were expressed per
gram of solids-not-fat.
No effects of vitamin and mineral supple-
ments were detected. The influences of age,
parity, and lactation history on milk compo-
sition are presented in Table 7. No differences
were observed in total solids. Milk from
women over 30 years old had a lower fat
content than that from women under 30.
Samples from women with additional chil-
dren had higher fat contents than those from
primiparae. Milk from older mothers had a
greater protein concentration than that from
younger mothers.
Mothers over 30 had increased copper

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80
U )
-J
a.
U )
66.
Ui
z
60
4 0
U )
Lii
-J
a.
.e
U )
Li.
0
Ui
z
20
120
80
60
40
20
0
FIG. 2. The distribution of iron in mature human FIG. I. The distribution of copper in mature human
milk.
i.Lg/mI
CU, FE, AND ZN IN MATURE HUMAN MILK
249
MEAN: 0.240.08
RANGE: 0.09-0.63
N : 350
contents per gram of milk solids. However,
this difference was not apparent when copper
was expressed per milliliter of milk or per
gram of solids-not-fat. W omen with addi-
tional children and lactation histories had
milk with higher copper contents than primi-
parous women. Iron content was lower in
older than in younger women. In contrast,
women with more than one child and lacta-
tion histories had a higher iron content than
primiparae. Increased age, multiparity, and
lactation history are all associated with in-
creased amounts of zinc in milk.
Discussion
As a basis for interpreting results, certain
characteristics of the subjects and the sam-
pling procedure should be considered. Sub-
jects volunteered to take part in this study;
therefore, willingness to provide samples be-
came a selection criterion along with ability
to lactate successfully. All analyses were
made on foremilk-the first milk secreted at
a feeding. It is generally agreed that fat
milk.
MEAN : 0.21 0.17
RANGE :(0I0 - .6
N : 350
content is at its lowest in foremilk and at the
early morning feeding when the majority of
samples were taken; therefore, data on fat are
not representative of the amount in total milk
secretion. Furthermore, the fact that the
caloric content of milk is largely dependent
on its fat content precludes the use of these
data for calculations of energy intakes.
Fat varied the most of all constituents
studied among women and among samples
from the same woman. Total solids patterns
reflected the wide variability of fat content.
Values ranged from 0.20 to 10.4 g/ 100 ml for
fat content and from 8.58 to 17.49 g/l00 ml
for total milk solids. The most striking obser-
ation was that during the within-day period,
80% of the total variance for fat content was
ascribable to variations among samples
within women, masking differences among
women. Kon and Mawson (21) observed that
the mean fat content was high when the
interval from the last feeding was short and
low when the interval was long. Since all
subjects fed their infants on demand, intervals
between feedings varied between 2 and 4 hr

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MEAN I 63 0.78
70
60
30
20
l0
U)
Ui
-J
a.
4
U )
Li.
0
Lii
D
z
milk.
TABLE 6
Comparison of morning, midday. and evening
mean values of milk constituents
0
FIG. 3. The distribution of zinc in mature human
11
250 PICCIANO AND GUTHRIE
Milk constituent
Time of day
Morning Mtdda Evening
Totalsolids(g/l00ml) ll.70ab l2.Sla l2.80b
Fat(g/lO0rnl) 2.96a 3.99a 4.Sla
Protein(g/lOOml) l.29a 1.30 l.3la
Copper
pg/mI milk 0.231 0.22 0.224
pg/g milk solids I .97ab I .84a I .Thb
pg/g solids-not-fat 2.68 2.70 2.76
Iron
pg/mI milk 0.l53ab 0.200a 0.21 lb
pg/g milk solids l.35ab l.61a l.67b
pg/gsolids-not-fat 2.59 2.38 1.79
Zinc
pg/mI milk l.7lab l.54a l.49b
pg/g milk solids l4.56ab l2.25a 11.6 lb
pg/g solids-not-fat 20.00 18.44 18.03
0 Means followed by the same letter arc significantI
different (P < 0.05).
during the day, possibly contributing to the
large within-day variations in fat content.
However, previous workers (2, 4, 24) have re-
ported variations equally as large throughout
the day, even when all of the milk was
sampled at scheduled intervals. Thus, most
investigators stress that a complete 24-hr
sample must be analyzed for a representative
estimate of fat content. These data clearly
support this conclusion.
Daily and weekly variations in fat content
can be attributable to individual differences
(58%). Even though all samples were ob-
tamed in the early morning at least 4 hr from
the last feeding, substantial variations (40%)
among samples within subjects were ob-
served. Large daily and weekly within-subject
variations suggest that even a 24-hr milk
sample may not be representative of the fat
content for an individual. In support of this
view, Salmi (25) found differences of about 2
g/ 100 ml for fat content of 24-hr samples in
each of his six subjects over a 14-day period.
Comparison of morning, midday, and eve-
ning values confirmed the work of early
investigators that milk fat is lowest in the
morning (2, 4). However, previous investi-
gators observed maximum values midmorn-
ing with a steady decline thereafter, in con-
trast to this study, where maximum values
occurred in the evening. A midmorning peak
could not have been detected, since no sam-
ples were taken at that time, whereas a high
evening value may have resulted from short
intervals between feedings.
Most of the variations that were observed
in protein content were attributable to indi-
vidual differences among subjects (95%).
W ith an average protein content of 1.28
g/lOO ml, values ranged from 0.76 to 2.04
g/ 100 ml. However, within an individual little
variation occurred. Although a slightly lower
protein concentration occurs in the morning
than at the evening feeding, it is not of a
magnitude to be nutritionally significant.
These observations are consistent with earlier
findings (4, 24) that one sample of human
milk from an individual would furnish a
representative estimate of protein content.
Before discussing general trends observed
for copper, iron, and zinc contents, considera-
tion must be given to the manner in which
these elements are expressed. Examination of
our results revealed that partitioned variances
for minerals expressed per gram of milk
solids often showed different patterns from
those expressed per milliliter of milk or per
gram of solids-not-fat. Data expressed on the

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CU, FE, AND ZN IN MATURE HUMAN MILK
251
bly be obtained from analysis of only one
TABLE 7
Influence of maternal age, parity, and lactation history
Milk constituent
A )
ge(yr
(no. of p
Parity
revious children)
Lactation history
(no. of times)
20-30 >30 0 lormore 0 lormore
Totalsolids(g/IOOml)
Fat(g/lOOml)
Protein(g/lOOml)
Copper (pg/mI)
Copper(pg/g milk solids)
Copper (pg/g solids-not-fat)
Iron(pg/ml)
Iron (pg/g milk solids)
Iron(pg/gsolids-not-fat)
Zinc(pg/ml)
Zinc(pg/g milk solids)
Zinc (pg/g solids-not-fat)
11.93
3.26
1.26
0.24
1.99
2.76
0.21
1.83
2.51
1.55
12.98
18.48
11.79
2.76#{176}
l.38C
0.25
2.lY
2.73
0.18#{176}
l.55c
2.00
2.00
17.02
20.98c
11.84
2.92
1.27
0.22
1.84
2.47
0.17
1.55
1.96
1.39
11.88
15.64
11.94
3.286
1.29
0.25c
2.lOi
2.89C
0.22C
l.89c
2.65
l.75c
14.57c
20.4 IC
11.87 11.93
3.03 3.26
1.29 1.27
0.23 0.25C
1.90 2.09C
2.57 2.89
0.17 0.2Y
1.54 l.94c
2.00 2.7lc
1.46 l.75c
12.38 14.59
16.67 20.39
0 <0.01. 6P <0.05.
C <0.001.
basis of milk solids may be misleading be-
cause milk solids contain the wide variability
of fat content. Thus, discussion of variation
patterns for copper, iron, and zinc is based on
analytical values expressed in terms of either
whole milk (micrograms per milliliter) or
solids-not-fat (micrograms per gram).
Copper content varied between 0.09 and
0.63 .ig/ml with a mean value of 0.24
g/ml. The frequency distribution of all
samples analyzed deviates from a normal
bell-shaped curve, with values skewed to the
right and a clumping of lower values. This
deviation from a normal distribution suggests
that there may be a physiological lower limit
of copper content. That is, the lower values
may represent the minimal amount of copper
that is present in human milk.
Among samples from the same individual,
copper varied more extensively over a period
of days and weeks than in a single day.
Variations in individuals for the within-day
period (12%) were of such a small magnitude
as to indicate that one sample of milk would
provide a representative estimate of copper
content on a particular day. However, parti-
tioning of variances for daily and weekly
samples showed that approximately 25% of
the total is attributable to variations within
individuals. Thus, it appears that several milk
samples taken over periods of days and weeks
are necessary to furnish a reliable estimate of
copper content for an individual.
Compared with copper, values for iron
exhibited a much wider range (<0.1 to 1.6
g/ml), while the mean value was similar
(0.21 zg/ml). Like copper, the frequency
distribution for all samples deviates from a
normal curve with skewing to the right. Thus,
the clustering of lower values may also be
interpreted to represent the minimal amount
of iron in human milk.
Analysis of weekly samples showed that
variations within individuals only accounted
for approximately 13% of the total variances,
implying that little variation in iron content
occurs with the progression of lactation. In
contrast, daily and within-day analyses illus-
trated that iron content varied considerably
among samples of individuals. In these
later periods, as much as 53% of observed
variance is ascribable to variations occurring
within individuals. Results of these analyses
are strongly suggestive that for a particular
individual one sample of milk does not pro-
vide a representative estimate of iron content.
Zinc content, with a mean value of 1.63
zg/ml, is considerably higher than either
copper or iron content. As can be seen from
the frequency histogram, values vary widely
from 0.14 to 3.95 g/ml. Unlike copper
and iron, zinc values are more evenly distrib-
uted over the reported range. The amount of
zinc present in human milk appears to be an
individual characteristic. Irrespective of ex-
perimental period, observed variance can be
almost totally accounted for by variations
among mothers (85%). Since variations
within mothers were of such a small magni-
tude (14%), a representative estimate of zinc
content in mature human milk could proba-

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252 PICCIANO AND GUTHRIE
sample. However, this has only been shown to
be valid over a 2-week period.
W hen comparisons of morning, midday,
and evening values were made, it was found
that copper and zinc contents were higher in
the morning while iron was lower at this time.
Since these differences were not evident when
minerals were expressed per gram of solids-
not-fat, it appears that fat content may have
an influence on the amounts of these elements
present in human milk. In bovine milk, the
largest part of both copper and zinc is bound
to the protein fraction (26, 27), whereas
only one-half of the iron is bound to this
fraction, the rest being associated with the
milk fat (28). Assuming similarity in
human milk, low-fat samples might be ex-
pected to contain less iron and more copper
and zinc than samples with a high fat content.
That this may be the situation is inferred
from these results since, as previously indi-
cated, milk fat is lowest in the morning.
No differences in the trace element con-
tent of milk from women receiving vitamin-
mineral preparations were noted even though
these women were receiving at least 30 mg of
iron/day. This finding is in agreement with
previous investigators who have been unsuc-
cessful in attempts to raise the iron level in
milk with dietary supplementation (29-31).
Likewise, there is no evidence to indicate that
the level of dietary copper influences the
amount in milk (32); however, Berfanstam
(33) was able to raise milk zinc content with
supplementation, suggesting that actual levels
may depend upon maternal intakes.
A recent investigation by Murthy and Rhea
(10) has provided data on copper, iron, and
zinc contents in 22 samples of milk from 13
women in the Cincinnati area. Mean copper
and zinc contents reported are essentially the
same as found in this study. This similarity of
values obtained from lactating women living
in two different geographic areas suggest that
there may be a homeostatic mechanism oper-
ating to maintain milk copper and zinc con-
tents within narrow limits. However, zinc
levels may only reflect a similarity in dietary
intakes. Mean iron content reported by these
investigators was 4 times greater than in our
study. It is not immediately apparent why
there is such a discrepancy with respect to
iron content. Their mean value is within our
observed range and possibly lack of agree-
ment is due to sample size differences.
The composition of milk is often linked
with the age of the mother and the number of
pregnancies and lactations she has ex-
perienced. However, these effects are usually
operating simultaneously because both multi-
parity and lactation history are related to
increased age. Although age- and parity-
related differences in fat content were ob-
served, it is not possible to determine whether
they are real or apparent because the samples
analyzed were not representative of fat con-
tent. Protein values were representative and
found to be higher in older than in younger
women. Recognizing that of the eight women
in this study over 30 years of age, two had
milk with a relatively high protein content,
this result may be an artifact of small num-
bers. After critically reviewing the literature
on this phenomenon, Morrison (29) postu-
lated that protein content of milk declines
with age. W ith respect to copper, iron, and
zinc contents, both multiparity and lactation
history appear to be associated with high
amounts. Also, higher copper and zinc con-
tents as well as a lower iron content were
found in older than in younger women. W hile
these data are suggestive that age, parity, and
lactation history exert an influence on copper,
iron, and zinc contents, a more reliable
indication of the effects of these factors would
probably require longitudinal rather than
cross-sectional data.
In view of the fact that human milk is still
considered the best food for the young infant,
it is believed that the assessment of the
nutritional requirements of infants can be
based on the composition of milk of well-
nourished mothers (21). Analyses were
made on 350 samples of milk from mothers
who were judged to be adequately nourished.
Since each woman provided seven samples,
average values for copper, iron, and zinc were
considered representative assuming that
amounts of elements in foremilk are repre-
sentative of the entire feed. Estimations of
infant intakes were calculated utilizing a milk
yield of 850 ml and an average infant weight
of 4 kg. From such calculations, it was
estimated that these fully breast fed infants
less than 3 months of age were ingesting
approximately 0.05 mg/kg per day of both

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CU, FE, AND ZN IN M A TU R E H U M A N M ILK 253
copper and iron and 0.35 m g/kg per day of
zinc. The Food and N utrition B oard pro-
posed that infants require betw een 0.05 and
0.1 m g/kg per day of copper and set the R D A
at 0.08 m g/kg per day (17). This recom -
m ended intake corresponds to a copper con-
tent of about 0.4 jzg/m l m ilk, a level found
in approxim ately 15% of our sam ples. The
recom m ended iron intake of 1.66 m g/kg per
day is about 30 tim es greater than that
received by our infants. For the totally breast
fed infant to m eet the R D A for iron, hum an
m ilk w ould have to contain 7 zg/m l, a
value considerably higher than any recorded
in this investigation. The recom m ended zinc
intake of 0.5 m g/kg per day w ould be m et if
infants consum ed hum an m ilk containing 3 to
4 zg/m l. O nly about 15% of our values
w ere in this range: therefore, the m ajority of
our infants w ere receiving less than the rec-
om m ended intake of zinc.
S ince m ost infants in the U nited S tates are
not breast fed (34, 35), it is of interest to
com pare the levels of copper, iron, and zinc in
hum an m ilk w ith those in cow s m ilk and
form ula preparations com m only used in in-
fant feeding. The range of reported values for
copper, iron, and zinc contents (m icrogram s
per m illiliter) of cow s m ilk are 0.05 to 0.30,
0.11 to 1.0, and 1.9 to 6.0, respectively (36,
37). Form ulas based on cow s m ilk w ere
found to contain from 0.13 to 0.25 g/m l
copper, 0.0 to 15.0 jzg/m l iron, and 1.8 to
3.5 zg/m l zinc. G enerally, soy-based for-
m ulas contained relatively high am ounts,
w ith values ranging (m icrogram s per m illi-
liter) from 0.32 to 0.5 for copper, 5.0 to 11.6
for iron, and 2.2 to 3.8 for zinc (38).
It is difficult to assess the adequacy of these
levels of trace elem ents because little is
know n about their availability. M oreover, it
is becom ing apparent that quantitative inter-
relationships am ong various trace elem ents
m ay play a m ore im portant role nutritionally
than the absolute am ount of a particular
elem ent. For exam ple, P riev (39) reported
that the am ount of iron assim ilated by the
new born is dependent upon the am ount of
copper in the diet. W iddow son and co-w ork-
ers (40) observed that full-term breast fed
infants retained m ore copper than preterm
infants given either cow s m ilk or a prepared
form ula w hich contained added copper m ak-
ing the concentration sim ilar to hum an m ilk.
These authors suggested that poor copper
retention in the preterm infants m ay have
resulted from high zinc intakes, a factor
know n to depress copper absorption. The
zinc to copper ratio of hum an m ilk is 6-8,
w hile it is 30-38 in cow s m ilk. H ow ever,
W alravens and H am bidge (41) recently re-
ported that infants fed a zinc-supplem ented
form ula (4 zg/m l) grew m ore rapidly and
had higher plasm a zinc levels than infants
fed the sam e form ula w ithout added zinc
(1.8 zg/m l). In this case, zinc supplem enta-
tion increased the zinc to copper ratio from 5
to 17. A dditional studies are obviously
needed to determ ine trace m ineral needs dur-
ing infancy.
Summar y
V alues for copper, iron, and zinc of 350
sam ples of m ature hum an m ilk from 50
w om en w ere found to vary betw een 0.09 and
0.63 tg/m l, <0.1 and 1.6 tg/m l, and
0.14 and 3.95 zg/m 1, respectively. R esults
from this study im ply that w ith respect to
copper and iron content, analysis of m ultiple
sam ples of m ilk from an individual is re-
quired to furnish a representative estim ate of
these trace elem ents. C onversely, only one
sam ple of m ilk from an individual m ay
furnish a representative estim ate of zinc con-
tent. R esults w ere also suggestive that age,
parity, and lactation history exert an influ-
ence upon am ounts of these trace elem ents
present. From these data on copper, iron, and
zinc content, it w as estim ated that fully
breast fed infants receive 0.35 m g/kg per day
of zinc and 0.05 m g/kg per day of both
copper and iron. fl
The authors gratefully acknow ledge the N ursing
M others C om m ittee of the C hildbirth E ducation A ssoci-
ation and La Leche League International for their help in
locating subjects and the cooperation of subjects m aking
t h i s s t u d y p o s s i b l e .
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