Variation of Superoxide Dismutase in Bovine Milk 1

J. HOLBROOK~ and C. L. HICKS

Department of Animal Science
University of Kentucky
Lexington 40506
ABSTRACT

that the enzyme may play an important role in

the oxidative stability of m i l l Superoxide dismutase catalyzes the dismutation of superoxide
anion as follows:

Individual cow milk is highly variable

in its resistance to oxidation. Variation
in the enzyme (superoxide dismutase)
concentration may have a minor effect on
the oxidative stability of individual cow's
milk. Milk from 11 Holsteins and 11 Jerseys were analyzed for 17 wk for superoxide dismutase, xanthine oxidase, oxidized lipids as determined by thiobarbituric acid values, milk produced, and fat
content. All cows were of good health
and on normal feed but varied in stage of
lactation and age. Average superoxide
dismutase concentration was 1.1 -+ .48
units/ml with a range of .06 to 2.88
units/ml and a variance of .23. Concentration of superoxide dismutase in milk was
approximately 102 lower than in bovine
blood. Assay of individual cow milk indicated no significant difference in superoxide dismutase concentration between
night and morning milkings. Also, superoxide dismutase concentration was affected by breed but not by stage of lactation
or age. Superoxide dismutase decreased
the minor pro-oxidant effect of xanthine
oxidase. However, the concentration of
superoxide dismutase in individual cow
milk did not account for the large variability in oxidative resistance in raw milk
that had not been exposed to light.

SOD -- Cu ++ + O~-aq -~ SOD -- Cu + + 02

SOD -- Cu + + O~-aq + 2H+ -* SOD -Cu ++ + H 2 0 2 ( 2 1 , 26)
Superoxide anions have been demonstrated in
milk illuminated with fluorescent light (22).
This is consistent with laboratory observations
of their presence with flavins and oxygen (13,
24, 28, 34). However, Aurand et aL (3) reported that flavin generated ringlet oxygen,
which is consistent with Type If, excited triplet
state flavin reactions (27, 35). Xanthine oxidase
(XO), a major enzyme in milk, generates superoxide anions under physiological conditions
(16, 26, 30). Breakdown of rhydroxyl peroxyl
radicals also may generate superoxide anions
(36). Deactivation of superoxide anions may
prevent their reaction with unsaturated fipids
(12, 30), although this reaction appears to be
unfavorable (19) compared to the Fenton-like
reaction (9) between superoxide anion and
hydrogen peroxide. Superoxide anion reacts
slowly with hydrogen peroxide to produce hydroxyl radicals (11, 20).
H 2 0 2 + O~--* OH" + O H - + 3 0 2

INTRODUCTION

Several workers (1, 14, 15, 22) have observed superoxide dismutase (SOD) (Erythrocuprin, 1.15.1.1) in bovine milk and suggested

If iron (Fe ++) is present, the reaction of superoxide anion with hydrogen peroxide is accelerated greatly by the following mechanism:
O~- + Fe +++ ~ Fe ++ + 3 02
F e ++

Received September 23, 1977.

t This m~nuscript (77-5-148) is published with the
approval of the Director of the Kentucky Agriculture
Experiment Station.
2Research and Development Department, Hawthorn Meilody, Inc., 4201 W. Chicago Avenue, Chi(:ago, IL 60651.

1978 J Dairy Sci 61:1072--1077

H 2 0 2 "* OH" + O H - + F e +++

(8, 2 0 )

Superoxide anion reduces the iron before it

reacts with H2 02 to produce the hydroxy radicals. Kellogg and Fridovich (19) suggested that
superoxide anion and hydrogen peroxide are

1072

SUPEROXlDE DISMUTASE IN MILK

necessary for rapid lipid oxidation, which may

indicate that hydroxyl radicals are responsible
for many of the oxidation problems in milk.
Hydroxyl radicals are highly reactive and probably generate other more stable radicals such as
carbonate radicals (9, 19) which initiate the
autooxidation mechanism.
Deactivation of superoxide anions and
hydrogen peroxide by SOD and catalase before
they react to generate highly activated oxygen
species is essential to preventing oxidation
problems under certain environmental conditions.
The purpose of this study was to determine
the amount of superoxide dismutase in milk
and how the concentration varied among
breeds, age, and stage of lactation. Also the
spontaneity of milk oxidation was evaluated
by co~relating the effect of SOD, XO, and fat
concentration with oxidation state as measured
by the thiobarbituric acid (TBA) assay.

MATER IALS A N D METHODS

Milk

Individual raw milk samples were collected

for 17 wk from 11 Holsteins and 11 Jerseys
from the University of Kentucky dairy herd.
Cows selected were free of mastiffs as determined by the California Mastiffs Test. No
cows had a high Cafiforuia Mastiffs score during
the experiment. Atl cows were on the same ration but varied in age and length of lactation.
Samples were collected and immediately cooled
to 4 C before being assayed.
Enzymes
Superoxide dismutase (bovine blood lot no.
16c-8030) containing 2880 U/rag solids, 2900
U/mg protein as assayed per McCorn and Fridorich (26) was purchased from Sigma Chemical
Company. Xanthine oxidase (lot no. 9034,
.514 U/m-g) was purchased from ICN Pharmaccuticals.
Ohemieal$
Cytochrome c (horse heart muscle lot no.
112647) was purchased from Aldrich Chemical
Company. All other chemicals were of analytical grade.

1073

Proeedures
Thiobarbituric acid values were determined
on 20 ml of milk by the procedure of Dunldey
and Jennings (10) in which the malonaldehyde
produced in the oxidation of lipids was measured spectrophotometrically.
Ten milliliters of the remaining samples were
centrifuged (20,000 rpm, 7.62 cm radius centrifuge head) for 1 hr at 7 C. The fat was suctioned from the remaining milk serum and pellet. The serum was removed and dialyzed in
simulated milk ultr~dtrate (17) for 24 h prior
to enzyme assay. Analysis of dialyzed solutions
minimized interference reactions caused by
ascorbic acid, tocopherols, and amino acids.
Xanthine oxidase was assayed spectrophotometrically by modifying the procedure of McCord and Fridovich (25) by adding I mM KCN
to inhibit the interference caused by SOD (2).
All assays were at 22 to 24 C, with .1 ml of
the dialyzed solution in the 3 ml reaction mixture. The reaction mixture contained 2 ml of
buffer (.015M sodium carbonate, pH 10, and
3.6 x 10-7M of cytochrome c), .1 ml of I mM
KCN, and .8 ml of distilled water. The reaction
rate was monitored against a fully reduced
reaction mixture in a Beckman DB spectrophotometer at 418 nm. Purified enzyme from
ICN Pharmaceuticals was used to prepare a
standard curve.
Superoxide dismutase assay was similar to
the XO assay and was based on the procedure
of Salin and McCord (32) in which SOD is detected by its ability to inhibit the reduction of
cytochrome c by XO. Due to the varying
amount of XO in each dialyzed milk serum
sample, the enzyme concentration was standardized arbitrarily in each reaction mixture to
1.38 x 10-2U. All assays were performed at 22
to 24 C, using .1 ml of the dialyzed solution in
a 2-ml reaction mixture. The reaction mixture
contained 2 ml of buffer (.015M sodium carbonate, pH 10, and 3.6 x 10-~M of cytochrome
c), and .9 ml of distilled water. The reaction
mixture was monitored the same as in the XO
procedure. Standard curves were prepared from
purified SOD purchased from Sigma Chemical
Company.
Experimental Design and Statistical Analysis

The experimental design was split plot hut

was not bah~ced for stage of lactation and age.
Journal of Dairy Science VoL 61, No. 8, 1978

1074

HOLBROOK AND HICKS

A large number of cows were in the first stage

of lactation early in the experiment but met the
requirements to be placed in the second stage
of lactation at the end of the experiment (Table
1). Also the greatest number of observations
were on cows which were 4 yr old (Table 2).
Therefore, means are adjusted for stage of
lactation and age, as well as individual cow and
breed differences. The statistical model in the
analysis was:

TABLE 2. Number of milk samples analyzed from

cows of different ages.
No. of
samples
15
40
103
44
71
30
16

No. of
cows

Age
of cows

3
7
3
5
2

3
4
5
6
7

Yijkl = / 2 + B i + C ( i ) j + A k + S 1 +
BAik + BSil + CS(i)jl + ASkl + 6(ijkl)
where
Y =

#=
Bi =

C(i)j =
Ak =
S1 =
6(ijkl) =

dependent variable
overall mean
effect of ith breed, i = 1, 2 (fixed)
effect of jth cow within ith breed,
j = 1 to 11 (random)
effect of kth age, k = 1 to 7 (random)
effect of lth stage of lactation, 1 =
1 to 3 (fixed)
within error (random)

Data were analyzed by regression procedure

outlined by the statistical analysis system of
Barr and Goodnight (SAS 72) (5).

milk since no additional activity was found

associated with the fat fraction. The concentration was similar to that found by Hill (15).
However, Asada (1) observed 5 U/mt in skim
milk. Differences in observed enzyme concentration probably relate to assay procedures used
and use of standard SOD sources. Although
SOD is in milk, its concentration is approximately 102 less than that in bovine blood.
Average XO concentration in milk serum
was 67.2 -+ 23.5 mUtml, with a variation of .55
mU/ml. Our values were similar to those reported by Cerbulis (6), whose total milk values
were 110 mU/ml, assuming 60% (29, 38) of the
XO was associated with the serum phase. Milk
contains approximately 50 times more XO than
SOD on a molar basis.

RESULTS AND DISCUSSION

Night and Morning Milkings
Enzyme Concentration

Superoxide dismutase and XO activities were

determined to quantify the relative amounts of
enzyme in the serum phase of bovine milk.
Average SOD concentrations from 319 observations was 1.12 -+ .48 U/ml. Superoxide
dismutase activity ranged from .06 to 2.88
U/ml, with a variance of .23 U/ml. Apparently
all of the SOD activity is in the serum phase of

Milk from night and morning milkings from

5 cows was analyzed for SOD concentration at
1 wk intervals for 6 wk. No significant change
in enzyme concentration was observed between the two milkings.
Effect of Breed

Adjusted mean differences due to breed are

in Table 3. Jersey milk contained more SOD
than Holstein milk, 1.27 U/ml, while Holstein
TABLE 1. Number of milk samples analyzed from milk averaged .92 U/ml.
cows at different stages of lactation.
Breed differences were signifcant for XO,
fat, and milk per milking (Table 3). Holsteins
No. of
No. of
Stage of
had lower XO and TBA in milk serum than Jersamples
cows
lactation
seys. The increase in XO concentration in Jersey milk might be expected since the enzyme is
60
4
0--100 days one of the major proteins in the fat globule
162
11
101-200
membrane. Milk from Jersey cows averaged
97
7
201-300
4.71% fat compared to 3.66% for Holstein
Journal of Dairy Science Vol. 61, No. 8, 1978

SUPEROXIDE DISMUTASE IN MILK

1075

TABLE 3. Effect of breed on superoxide dismutase, xanthine oxidase, thiobarbituric acid, milk, and fat. a

Breed

Superoxide
dismutase* *

Holstein
Jersey

.92
1.27

Xanthine
oxidase* *

Thiobarbituric
acid values

Kg milk/
milkings*

%
Fat* *

.0605
.0775

.038
.034

21.4
17.0

3.66
4.71

(units/ml)

aMeans adjusted for cows within a breed and stage of lactation.

*P<.05.
**P<.01.

cows.
Effect of Lactation and Age

Stage of lactation was divided into three

intervals so that adjusted mean data could be
presented (Table 4). Milk per milking and TBA
values decreased significantly as the stage of lactation progressed. The changes of milk per
milking and fat concentration as affected by
stage of lactation and season were similar to
those in (18). The decrease in TBA values,
which is a measure of the milk's oxidative stability, was similar to Kratzers' (23) observation that milk becomes more resistant to oxidation as stage of lactation increases.
Xanthine oxidase and SOD concentration
did not change significantly with stage of lactation. Zikakis et al. (37) observed a decrease in
XO in human milk whereas Rajan (31) observed
a substantial increase in XO in bovine milk with
increasing lactation time. Apparently other fac-

tors are involved which affect the XO concentration in milk. Cows is this study were selected
on low cellular counts in their milk as detected
by the California Mastitis Test (CMT). No cows
had high CMT scores (less than 1) during our
study. Cone (7) observed that milk had increasing somatic cell counts as the stage of lactation
progressed. This increase in somatic ceils may
be responsible for the increased XO activity
that was observed by Rajan (31). Cow age had
no significant effect on observed values for
enzyme concentration or TBA.
Effect of Enzyme Concentration on Oxidation

A significant increase in SOD activity

(P>.0004) and TBA values (P>.0108) was observed in milks that contained high XO concentrations, as given by the following regression
equation:
X O c o n c . = . 0 5 6 5 + . 0 1 0 2 x t +.0820x2

TABLE 4. Effect of stage of lactation on superoxide dismutase, xanthine oxidase, thiobarbituric acid, milk, and
fat. a

Stage of
lactation

Superoxide
dismutase

(days)

0-100
101-200
201-300

1.05
1.08
1.17

Xanthine
oxidase

Thiobarbituric
acid values* *

Kg milk/
milking* *

%
Fat*

.078
.066
.063

.078
.037
-.003

22.7
18.1
16.1

4.14
4.10
4.27

(units/ml)

aMeans were adjusted for cows within a breed and breed.

*P<.05.
**P<.O1.
Journal of Dairy Science Vol. 61, No. 8, 1978

1076

HOLBROOK AND HICKS

w h e r e x l = SOD c o n c e n t r a t i o n a n d x2 = T B A

ACKNOWLEDGMENTS

value. The increase in TBA values might be exThis work was supported in part b y a grant
pected since XO was a pro-oxidant (16, 26, 30). from Dairy Research Incorporated.
Aurand (4) first reported XO was a pro-oxidant
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Journal of Dait3" Science Vol. 61, No, 8, 1978