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Copper Distribution in Milk during Early Lactation1
COPPER DISTRIBUTION
IN MILK DURING EARLY LACTATION 1
R. L. K I N G ANn W. F. WILLIAMS Department of Dairy Science, University of Maryland, College Park
SUM:MARY
Cue4 was used to detelm~ine the distribution of natural copper in milk at intervals during early lactation. The amount of copper associated with fat globules was about 15% after 2 and 4 wk of lactation and 35% after 10 wk. The change in copper distribution was accompanied by a change in susceptibility to oxidized flavor. A combination of cream washing and chelation by EDTA removed u p to 90% of lipid-bound copper. When these treated fat globules were added back to and recovered from the original skimmilk, their copper content increased. Administration of Cu ~ by infusion or by drench yielded similar results with respect to distribution, but rate of appearance in milk was slower by drenching. presented represent the milk produced 24 hr after administration of the isotope. Radioactivity was determined o n 2-ml samples using a 2-in. Na I (T1.) well crystal detector. Copper removal. Cream obtained from one lot of milk was washed one time with four volumes 40 C tap water to remove the majority of the skimmilk, and divided into two lots. EDTA (disodinm salt) was added to one lot at a concentration of 2 g/l,000 g cream. After 2 hr at room temperature, both lots were washed an additional two times and standardized to the same fat content. A portion of the EDTA-treated washed cream was added back to the original skimmilk to yield a reconstituted milk having the same fat content as the original milk. After 2 hr at room temperature the reconstituted milk was separated and the cream washed three times, thus making a total of six washings for this lot of fat globules. The radioactivity of the various fractions was determined.
The incidence of spontaneous oxidized flavor was found to be correlated with the natural copper content of the milk during periods when the cows were on dry rations (1). A n exception to this relation was found in milks produced during very early lactation. Though quite high in copper content (1, 4), these milks were generally resistant to oxidized flavor (1). Similarly, butter produced from such milk was resistant to oxidative defects during storage (5). I t has been suggested that the distribution of copper in milk is an important factor in determining the susceptibility to oxidized flavor (3). The present study was designed to determine the distribution of copper in milk produced during the initial stage of lactation and the effect, if any, on the flavor stability. Some effects concerning the chemical association of copper were also observed. EXPERII~EI~TAL PROCEDURE
Holsteins, selected from the University dairy herd, were given Cu u by a slow infusion technique as previously described (3). I n one experiment the isotope 2 was administered directly into the rumen with a tygon tube inserted through the nostril. The entire milk from a single milking was fractionated by methods previously described (3). The data
RESULTS AI~IDDISCUSSIOI~ The distribution of natural copper in a milk produced 2 wk after parturition is shown in Table 1. The activity of the washed milk represents copper associated with the fat phase and indicates that this phase is much less rich in copper than the fat phase of a spontaneous milk which contained over 35% of the total activity (3). The difference is also apparent when the activity is related to the protein content of the various fractions. F o r a given concentration of protein, the membrane material of this early lactation milk contained about 12 times as much copper as the other proteincontaining fractions; whereas, in the spon-
Received for publication October 4, 1962. ~Seientifie Article ~o. A10.08. Contribution 1~o. 3396 of the 1VIaryland Agricultural Experiment Station, Department of Dairy Science. 2C.u~ was obtained from the Oak Ridge National Laboratory as cupric nitrate. Approximately 20 n ~ were infused per experiment. 11
R. L. K I N G A N D W. F. W I L L I A M S
]2
TABLE 1 Distribution of natural copper in milk 2 wk post-partum
Sample description
Activity
(count/rain) Whole milk Skimmilk Washed milk " Isoelectrlc casein Isoelectrie whey Membrane solution b
819 694 122 477 208 836
Activity of whole milk
Activity per 1% of protein
( %)
(count/rain)
100 84.8 14.9 58.3 24.4 ......
283 222 215 224 2,785
~4 × washed cream diluted to same fat content as whole milk before counting. Obtained by filtering churned, washed cream. taneous milk, copper was 50 times more concentrated in the membrane fraction than in the other fractions. The casein and whey fractions were about equal in copper concentrat i o n / u n i t of protein. The effect of increasing stage of lactation on the distribution of natural copper in milk f r o m a single cow is shown in Table 2. The milk produced a f t e r 4 wk of lactation had a distribution of copper between the f a t globules and skimmilk similar to the milk produced a f t e r 2 wk of lactation f r o m a different animal (Table 1). A f t e r 10 wk of lactation the distribution was markedly different and v e r y similar to that found f o r a spontaneous-type milk (3). The milk produced a f t e r 10 wk of lactation was not spontaneous, but was susceptible to oxidized flavor. The milks produced a f t e r 2 and 4 wk of lactation were resistant to oxidized flavor (2). The copper content of the 4-wk-laetation milk was 0.094 p p m and a f t e r 10 wk it was 0.055 p p m . The distribution of added copper in these early lactation milks was similar to that reported f o r normal milk with less than 3 % associated with the f a t globules.
Results obtained in the comparison of drenching versus infusion of Cu ~ on the distribution of copper in milk were something less than conclusive. No marked differences were app a r e n t ; however, the activity of the milk obtained a f t e r drenching was quite low (less than 100 counts/min) and the cow had a severe case of mastitis the following week when the infusion trial was conducted. One marked difference observed between the two methods was in the rate of appearance of activity in the milk. W h e n the isotope was infused, the first milk (8-12 h r a f t e r infusion) contained nearly twice the activity of the second milk (24 h r a f t e r infusion). W h e n the isotope was given as a single drench, the activity of the second milk was over three times that of the first milk. Similar observations were r e p o r t e d when the copper content of milk was increased by drenching with CuSO, (1). N a t u r a l copper in milk is not dialyzable even when the p H is lowered to 3 ( 3 ) ; however, it does become dialyzable when E D T A is added to milk (2). I n the present study, it was found that about 90% of natural copper associated with washed f a t globules could be
TABLE 2 Effect of increasing stage of lactation on distribution of natural copper in milk 4 wk
Sample description Whole milk Skimmilk Washed milk a Isoelectric casein Isoeleetric whey Membrane solution b
10 wk
Activity
Activity of whole milk
Activity
(count/rain)
(%)
(count/rain)
1,955 1,698 298 1,274 290 3,949
100 86.8 15.2 65.3 14.8 ......
2,128 1,304 735 1,072 330 9,982
Activity of whole milk
(%) 100 61.3 34.9 50.4 15.5 ......
a4 × washed cream diluted to same fat content as whole milk before counting. b Obtained by iiltering churned, washed cream.
COPPER DISTt%IBUTION IN MILK
TABLE 3 Effect of E D T A on the distribution of natural copper in milk Saznple description
Activity
(eount/mi~ ) Original milk Original skimmilk Reconstituted milk Reseparated skimmilk 3 × washed cream ~ 3 × washed cream -}- E D T A 6 × washed cream from reconstituted milk
819 694 657 668 877 1~8 168
All washed cream standardized to 35% fat. r e m o v e d by t h e a d d i t i o n of E D T A , followed b y several w a s h i n g s to remove the chelated c o p p e r a n d excess E D T A . W h e n the c o p p e r p o o r f a t globules were a d d e d b a c k to t h e orig'inal skimmilk a n d t h e n r e s e p a r a t e d a f t e r 2 h r time, t h e i r activity increased a n d the a c t i v i t y of the skimmilk decreased b y a c o m p a r a b l e m a g n i t u d e . These results are s h o w n in T a b l e 3 a n d indicate t h a t a n e q u i l i b r i u m w i t h respect to c o p p e r m a y exist between the v a r i o u s copp e r - c o n t a i n i n g substances i n m i l k ; or conversely, t h a t the c o p p e r b i n d i n g activity o f the f a t globule m e m b r a n e is g r e a t e r t h a n t h a t of
13
some o t h e r c o p p e r - b i n d i n g s u b s t a n c e in skimmilk. This is c o n s i s t e n t w i t h the m u c h g r e a t e r c o n c e n t r a t i o n of n a t u r a l c o p p e r in association w i t h the f a t globule m e m b r a n e m a t e r i a l as c o m p a r e d to the o t h e r p r o t e i n - c o n t a i n i n g f r a c tions of milk. REFERENCES (1) KING, R. L., AND DUIqKL]~Y~ W. L.
Relation
of Natural Copper in Milk to Incidence of Spontaneous Oxidized Flavor. J. Dairy Sci., 42: 429. 1959. (2) KING, R. L., AND D U N K L ~ W. L. Role of a Chelating Compound in the Inhibition of Oxidized Flavor. J. Dairy Sci., 42: 897. 1959. (3) KING, R. L., LUICK, J. R., LITM~',r, I. I., JENNINGS, W.
G., AN]) DUNKL~f~ W. L.
Distribution of Natural and Added Copper and Iron in Milk. J. Dairy Sci., 42: 780. 1959. (4) KOPPFJJAN, C. A., AND MULD~E~,, H .
T h e Cop-
per Contents of Milk. Prec. 13th Intern. Dairy Congr., 3: 1400. 1953. (5) ME~GE~, J. W. The Influence of Copper, Iron and Manganese on the Development of Coldstorage Defects in Butter. Mcdedel. Landbouwhooge-school Wageningen, 61: 1. 1961. [Cited from Dairy Sci. _&bstrs., 24: 105. 1962.]
IN MILK DURING EARLY LACTATION 1
R. L. K I N G ANn W. F. WILLIAMS Department of Dairy Science, University of Maryland, College Park
SUM:MARY
Cue4 was used to detelm~ine the distribution of natural copper in milk at intervals during early lactation. The amount of copper associated with fat globules was about 15% after 2 and 4 wk of lactation and 35% after 10 wk. The change in copper distribution was accompanied by a change in susceptibility to oxidized flavor. A combination of cream washing and chelation by EDTA removed u p to 90% of lipid-bound copper. When these treated fat globules were added back to and recovered from the original skimmilk, their copper content increased. Administration of Cu ~ by infusion or by drench yielded similar results with respect to distribution, but rate of appearance in milk was slower by drenching. presented represent the milk produced 24 hr after administration of the isotope. Radioactivity was determined o n 2-ml samples using a 2-in. Na I (T1.) well crystal detector. Copper removal. Cream obtained from one lot of milk was washed one time with four volumes 40 C tap water to remove the majority of the skimmilk, and divided into two lots. EDTA (disodinm salt) was added to one lot at a concentration of 2 g/l,000 g cream. After 2 hr at room temperature, both lots were washed an additional two times and standardized to the same fat content. A portion of the EDTA-treated washed cream was added back to the original skimmilk to yield a reconstituted milk having the same fat content as the original milk. After 2 hr at room temperature the reconstituted milk was separated and the cream washed three times, thus making a total of six washings for this lot of fat globules. The radioactivity of the various fractions was determined.
The incidence of spontaneous oxidized flavor was found to be correlated with the natural copper content of the milk during periods when the cows were on dry rations (1). A n exception to this relation was found in milks produced during very early lactation. Though quite high in copper content (1, 4), these milks were generally resistant to oxidized flavor (1). Similarly, butter produced from such milk was resistant to oxidative defects during storage (5). I t has been suggested that the distribution of copper in milk is an important factor in determining the susceptibility to oxidized flavor (3). The present study was designed to determine the distribution of copper in milk produced during the initial stage of lactation and the effect, if any, on the flavor stability. Some effects concerning the chemical association of copper were also observed. EXPERII~EI~TAL PROCEDURE
Holsteins, selected from the University dairy herd, were given Cu u by a slow infusion technique as previously described (3). I n one experiment the isotope 2 was administered directly into the rumen with a tygon tube inserted through the nostril. The entire milk from a single milking was fractionated by methods previously described (3). The data
RESULTS AI~IDDISCUSSIOI~ The distribution of natural copper in a milk produced 2 wk after parturition is shown in Table 1. The activity of the washed milk represents copper associated with the fat phase and indicates that this phase is much less rich in copper than the fat phase of a spontaneous milk which contained over 35% of the total activity (3). The difference is also apparent when the activity is related to the protein content of the various fractions. F o r a given concentration of protein, the membrane material of this early lactation milk contained about 12 times as much copper as the other proteincontaining fractions; whereas, in the spon-
Received for publication October 4, 1962. ~Seientifie Article ~o. A10.08. Contribution 1~o. 3396 of the 1VIaryland Agricultural Experiment Station, Department of Dairy Science. 2C.u~ was obtained from the Oak Ridge National Laboratory as cupric nitrate. Approximately 20 n ~ were infused per experiment. 11
R. L. K I N G A N D W. F. W I L L I A M S
]2
TABLE 1 Distribution of natural copper in milk 2 wk post-partum
Sample description
Activity
(count/rain) Whole milk Skimmilk Washed milk " Isoelectrlc casein Isoelectrie whey Membrane solution b
819 694 122 477 208 836
Activity of whole milk
Activity per 1% of protein
( %)
(count/rain)
100 84.8 14.9 58.3 24.4 ......
283 222 215 224 2,785
~4 × washed cream diluted to same fat content as whole milk before counting. Obtained by filtering churned, washed cream. taneous milk, copper was 50 times more concentrated in the membrane fraction than in the other fractions. The casein and whey fractions were about equal in copper concentrat i o n / u n i t of protein. The effect of increasing stage of lactation on the distribution of natural copper in milk f r o m a single cow is shown in Table 2. The milk produced a f t e r 4 wk of lactation had a distribution of copper between the f a t globules and skimmilk similar to the milk produced a f t e r 2 wk of lactation f r o m a different animal (Table 1). A f t e r 10 wk of lactation the distribution was markedly different and v e r y similar to that found f o r a spontaneous-type milk (3). The milk produced a f t e r 10 wk of lactation was not spontaneous, but was susceptible to oxidized flavor. The milks produced a f t e r 2 and 4 wk of lactation were resistant to oxidized flavor (2). The copper content of the 4-wk-laetation milk was 0.094 p p m and a f t e r 10 wk it was 0.055 p p m . The distribution of added copper in these early lactation milks was similar to that reported f o r normal milk with less than 3 % associated with the f a t globules.
Results obtained in the comparison of drenching versus infusion of Cu ~ on the distribution of copper in milk were something less than conclusive. No marked differences were app a r e n t ; however, the activity of the milk obtained a f t e r drenching was quite low (less than 100 counts/min) and the cow had a severe case of mastitis the following week when the infusion trial was conducted. One marked difference observed between the two methods was in the rate of appearance of activity in the milk. W h e n the isotope was infused, the first milk (8-12 h r a f t e r infusion) contained nearly twice the activity of the second milk (24 h r a f t e r infusion). W h e n the isotope was given as a single drench, the activity of the second milk was over three times that of the first milk. Similar observations were r e p o r t e d when the copper content of milk was increased by drenching with CuSO, (1). N a t u r a l copper in milk is not dialyzable even when the p H is lowered to 3 ( 3 ) ; however, it does become dialyzable when E D T A is added to milk (2). I n the present study, it was found that about 90% of natural copper associated with washed f a t globules could be
TABLE 2 Effect of increasing stage of lactation on distribution of natural copper in milk 4 wk
Sample description Whole milk Skimmilk Washed milk a Isoelectric casein Isoeleetric whey Membrane solution b
10 wk
Activity
Activity of whole milk
Activity
(count/rain)
(%)
(count/rain)
1,955 1,698 298 1,274 290 3,949
100 86.8 15.2 65.3 14.8 ......
2,128 1,304 735 1,072 330 9,982
Activity of whole milk
(%) 100 61.3 34.9 50.4 15.5 ......
a4 × washed cream diluted to same fat content as whole milk before counting. b Obtained by iiltering churned, washed cream.
COPPER DISTt%IBUTION IN MILK
TABLE 3 Effect of E D T A on the distribution of natural copper in milk Saznple description
Activity
(eount/mi~ ) Original milk Original skimmilk Reconstituted milk Reseparated skimmilk 3 × washed cream ~ 3 × washed cream -}- E D T A 6 × washed cream from reconstituted milk
819 694 657 668 877 1~8 168
All washed cream standardized to 35% fat. r e m o v e d by t h e a d d i t i o n of E D T A , followed b y several w a s h i n g s to remove the chelated c o p p e r a n d excess E D T A . W h e n the c o p p e r p o o r f a t globules were a d d e d b a c k to t h e orig'inal skimmilk a n d t h e n r e s e p a r a t e d a f t e r 2 h r time, t h e i r activity increased a n d the a c t i v i t y of the skimmilk decreased b y a c o m p a r a b l e m a g n i t u d e . These results are s h o w n in T a b l e 3 a n d indicate t h a t a n e q u i l i b r i u m w i t h respect to c o p p e r m a y exist between the v a r i o u s copp e r - c o n t a i n i n g substances i n m i l k ; or conversely, t h a t the c o p p e r b i n d i n g activity o f the f a t globule m e m b r a n e is g r e a t e r t h a n t h a t of
13
some o t h e r c o p p e r - b i n d i n g s u b s t a n c e in skimmilk. This is c o n s i s t e n t w i t h the m u c h g r e a t e r c o n c e n t r a t i o n of n a t u r a l c o p p e r in association w i t h the f a t globule m e m b r a n e m a t e r i a l as c o m p a r e d to the o t h e r p r o t e i n - c o n t a i n i n g f r a c tions of milk. REFERENCES (1) KING, R. L., AND DUIqKL]~Y~ W. L.
Relation
of Natural Copper in Milk to Incidence of Spontaneous Oxidized Flavor. J. Dairy Sci., 42: 429. 1959. (2) KING, R. L., AND D U N K L ~ W. L. Role of a Chelating Compound in the Inhibition of Oxidized Flavor. J. Dairy Sci., 42: 897. 1959. (3) KING, R. L., LUICK, J. R., LITM~',r, I. I., JENNINGS, W.
G., AN]) DUNKL~f~ W. L.
Distribution of Natural and Added Copper and Iron in Milk. J. Dairy Sci., 42: 780. 1959. (4) KOPPFJJAN, C. A., AND MULD~E~,, H .
T h e Cop-
per Contents of Milk. Prec. 13th Intern. Dairy Congr., 3: 1400. 1953. (5) ME~GE~, J. W. The Influence of Copper, Iron and Manganese on the Development of Coldstorage Defects in Butter. Mcdedel. Landbouwhooge-school Wageningen, 61: 1. 1961. [Cited from Dairy Sci. _&bstrs., 24: 105. 1962.]