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A Study of Variations in Secretion of Ca45 by the Mammary Glands of Dairy Cows1, 2
A STUDY
OF VARIATIONS
IN SECRETION
OF
Ca ~5 B Y T H E
M A M M A R Y G L A N D S O F D A I R Y C O W S 1,2 E. W. SWANSON, R. A. MONROE, 3 D. B. ZILVERSMIT,~ W. J. VISEK, 5 AND C. L. COMAR~
Depart~aent of Dairying, University of Tennessee, Knoxville, and Ato,nic Energy Commission Agric,~dtural Research Program, Oat~ Ridge, Tennessee
A better understanding of the causes of low blood calcium after parturition is important for proper control of milk fever. This series of experiments has helped clarify some of the questions regarding calcium metabolism, though no satisfactory explanation for the excessive proportion of radioactive calcium in milk has been found. E d i t o r .
The c a l c i u m m e t a b o l i s m of the l a c t a t i n g d a i r y cow proceeds at a h i g h r a t e because of the secretion into m i l k of 20 to 30 g. of c a l c i u m each day. This is a b o u t f o u r times the c a l c u l a t e d fecal metabolic loss of c a l c i u m b y the cow (16). B a l a n c e s t u d i e s have shown t h a t the l a c t a t i n g cow is n o r m a l l y i n n e g a t i v e c a l c i u m b a l a n c e i n the m a j o r p a r t of the l a c t a t i o n period, even w h e n a d e q u a t e l y fed (3). The f u n c t i o n i n g u d d e r has been s h o w n to be the cause of drastic c h a n g e s i n c a l c i u m c o n t e n t of the blood at the i n i t i a t i o n of l a c t a t i o n (12), a n d d a i r y cows are u n i q u e l y afflicted with a n a i l m e n t , m i l k fever, caused b y low blood c a l c i u m a f t e r p a r t u r i t i o n (8). A n u n d e r s t a n d i n g of the secretion of milk c a l c i u m a n d its p r o b a b l e p r e c u r s o r forms a n d sources has b e e n s o u g h t i n previous s t u d i e s b y the use of r a d i o a c t i v e c a l c i u m b y which blood c a l c i u m a n d feed c a l c i u m have been l a b e l e d (15). These s t u d i e s i n d i c a t e d t h a t some cows a p p a r e n t l y were able to c o n c e n t r a t e r a d i o a c t i v e c a l c i u m i n t h e i r m i l k a f t e r oral d o s i n g to a g r e a t e r e x t e n t t h a n t h e y c o n c e n t r a t e d stable calcium, a l t h o u g h this p h e n o m e n o n was n o t so n o t i c e a b l e a f t e r i n t r a v e n o u s dosing. A f t e r both oral a n d i n t r a v e n o u s doses, the p r o p o r t i o n of r a d i o a c t i v e to stable c a l c i u m r e m a i n e d h i g h e r in milk t h a n i n blood, even a f t e r c o r r e c t i o n for time of m i l k secretion. Si,we this difference in the p e a k specific a c t i v i t y of blood a n d m i l k c a l c i u m was Received for publication March 30, 1956. 1 This manuscript is published with the permission of tile Director of the University of "remlessee Agricultural Experiment Station, Knoxville. 2 This work was completed under Contract No. AT-40-1-GEN-242 between the University of Tennessee, College of Agriculture, and the Atomic Energy Commission. The radioactive materials used in this work were obtained from the Oak Ridge National Laboratory on allocation from the United States Atomic Energy Commission. Present address: University of California, Atomic Energy Project, West Los Angeles. 4 Present address: Department of Physiology, University of Tennessee, l~Iemphis. Present address: University of Chicago, Medical School. " Present address: Oak Ridge Institute of Nuclear Studies, Medical Division, Oak Ridge. 1594
SECRETION
OF Ca 4n B Y T H E
51AMMARY G L A N D S
1595
at variance with the expected precursor-product relationship (18), the additional studies presented in this article were coudueted to seek a f u r t h e r explanation for this phenomenon. MATERIALS
AND
METHODS
The cows used in this study either were all Jerseys or carried at least 50% J e r s e y blood and were mature in age. They varied widely in milking ability and stage of lactation through the series of experiments. They were fed a medium to poor quality mixed hay and commercial 16% protein dairy feed which was fortified with mineral supplements containing adequate calcimn, phosphorus, and minor elements, l)osing, sampling, and analyses were done by the methods previously described (2, 15). Intravenous injection of 10 units oxytocin was given prior to all milkings during the first 2 days of each experiment and for all later periods in which frequent milkings were made. Special methods for individual experiments are described below with the discussion of those experiments. Specific activity of calcium has been calculated as the percentage of the dose of Ca ~5 times 10 * per milligram of total calcium. In plotting the specific activities of calcium from milk and urine, the median time between sample collections has been used r a t h e r than the time of collection. This correction for time of secretion of milk was assumed to be adequate because it was f o u n d that the specific activities of first and last drawn milk a f t e r oxytocininduced milk ejection at one milking period differed only slightly, being 1.93 and 1.83, respectively. All changes in specific activity with time have been plotted on semilogarithmic scales, according to time after dosing. RESULTS
Comparing oral and intravenous doses. Since comparisons of specific activity of blood and milk calcium after oral and intravenous doses in the same cow had not been presented previously, these data were obtained from two cows in mid-lactation. They were given oral doses of approximately 5 inc. Ca45C12 December 14, 1951, and F e b r u a r y 1, 1952. They were dosed intravenously with 1.75 inc. of Ca45C12 ~[arch 5, 1952. The results of these trials with cow 1 are shown in Figures I to 3. V e r y similar results were obtained in the trials with cow 5. These figures show that the declining curve of blood calcium specific activity a f t e r an intravenous dose passed through the peak of the time-corrected milk curve, but later a slight delay in calcium secretion into milk was indicated. In the straight declining phase of the curves the blood and milk values were parallel b.~lt separated by a time factor of about 8 hours. These differences might be explained by a calcium reservoir in the udder requiring an average of 8 hours for milk precursor calcium to traverse. The specific activity curves a f t e r oral doses were somewhat different. In the December trials the peaks of milk and blood calcium specific activity were nearly equal, but in the F e b r u a r y £rials milk reached a higher specific activity than blood, as had been noted
1596
E. ~V. SWANSON- ET AL
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]~m. 1. C o m p a r i s o n of the specific act i v i t y of blood a n d m i l k c a l c i u m a f t e r an o r a l dose of Ca ~ g i v e n to cow I D e c e m b e r 14, 1951.
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Fro. 3. C o m p a r i s o n of the specific act i v i t y of blood a n d m i l k c a l c i u m a f t e r all i n t r a v e n o u s dose of Ca ~ g i v e n to cow 1 M a r c h 5, 1952.
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FIG. 4. C o n l p a r i s o n of t he specific act i v i t y of blood a n d m i l k c a l c i u m a f t e r a n oral dose of Ca 45 g i v e n to cow 5 w i t h collec t i ons of s a m p l e s a t 4 to 6 h m l r i n t e r v a l s .
before. The straight declining blood and milk curves for cow 1 were nearly parallel, but for cow 5 they seemed to diverge in later samplings. The time differences between the curves for cow I were 13 and 17 hours and those for cow 5 were 14 to 20 hours and 20 to 26 hours, respectively, in the two trials. I f the u d d e r were p r e f e r e n t i a l l y concentrating radioactive calcium, which after an oral dose is a representative tracer of feed calcium, the specific activity decline curves of blood and milk would be expected to diverge. The observation
SECRETION OF Ca~ BY TItE MAMMARYGLANDS
1597
that they diverged with one cow and not with the other, irrespective of tile relation of milk and blood calcium specific activities at the peaks, makes the i n t e r p r e t a t i o n of this phenomenon difficult. These types of inconsistencies have a p p e a r e d at other times in these studies. There was a strong suggestion, however, t h a t the cow's m a m m a r y gland m a y handle orally derived calcium differe n t l y f r o m t h a t placed into the blood stream all at one time. Blood and milk calcium specific activity changes after oral doses. The possibility of the blood calcium reaching a higher peak specific activity t h a n t h a t of milk a f t e r an oral dose, but m a i n t a i n i n g it for so short a time that it was missed in the sampling schedule, was investigated with two cows f r o m which v e r y frequent samples were taken in the early phase of the trial. E a c h cow was dosed orally with about 5 inc. of Ca4~Cl~o. I f the contents of the dosing capsule were not thoroughly mixed in the cow's stonlach but were r a p i d l y shunted to an area where the m a j o r portion was absorbed, such a t r a n s i t o r y peak blood specific activity would be possible. 5.0
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]~m. 5. Comparison of the specific gravity of blood and milk calcium after an oral dose of Ca~ given to cow 11 with frequent collection of samples. The results of these trials are shown in F i g u r e s 4 and 5. E v e n with samples t a k e n at 4-hour intervals, the specific activity of none of the blood samples f r o m cow 5 was as high as t h a t of the seven highest milk samples ( F i g u r e 4). The peak specific activity of the blood was not attained until 34 hours a f t e r dosing, which indicates a r a t h e r slow absorption of the dose. The peak specific activity a p p e a r e d in the milk at about the same time as the blood peak. I n cow 11, however, peak specific activity was f o u n d in the 12-hour blood samples, which exceeded the highest specific activity f r o m a n y of the milk samples (Figure 5). The peak for specific activity in milk occurred at about 20 hours. These
1598
E.w.
SW.(NSON ET .(L
two cows clearly absorbed and metabolized their ingested calcium at different rates. Cow 11, which absorbed the dose more rapidly, failed to concentrate the radioactive calcium in the milk as did cow 5, which absorbed the dose more slowly. The specific activity curves of the blood and milk of both cows appeared to diverge slightly in the declining or p r e s u m a b l y post-absorptive phase. These first experiments seemed to confirm the previous observation (15) that the mamm a r y gland was concentrating radioactive calcium in preference to stable calcium a f t e r an oral dose in some cows at some periods; but the effect was not always observed.
Test for a high specific activity blood fraction by electrophoresis after adding Ca ''~. Although Visek et al. (15) had not been able to find blood fractions that differed significantly in specific activity f r o m the whole blood calcium by means of ultrafiltration and adsorption, the possibility t h a t serum calcium is not all of equal mobility still exists. One example which m i g h t be due to differences in mobility of radioactive calcium in the bloocl is the observation that specific activity of blood calcium of the new-born calf f r o m a cow dosed before p a r t u r i tion was 2.5 times t h a t of the cow (15). A f u r t h e r a t t e m p t to detect differences iu blood calcium fractions was made by a combination of p a p e r electrophoresis and autoradiographic techniques. The method of Gordon et al. (4) was used. All strips were moistened with 0.05 M veronal buffer ( p H 8.5) and 5 to 10 )t of sample were applied to the moist strip before a potential gradient of 5 volts per centimeter was established. The results of three typical tests arc shown in F i g u r e 6. Strip a is an autor a d i o g r a m of a sample of Ca4"~C12 a f t e r 1 hour electrophoresis in veronal containing 0.9% NaC1. All of the radioactive calcium had m i g r a t e d a distance of 7 cm. Strips b and c resulted f r o m the same t r e a t m e n t of bovine serum, which had been incubated with Ca 4n. The a u t o r a d i o g r a m is superimposed upon the p a p e r strip, which was stained with bromphenol blue to show the proteins. I t is evident t h a t the radiocalcium had m i g r a t e d the same distance as the calcium chloride (strip a) and t h a t it had separated cleanly f r o m the plasma proteins except for a few intensely radioactive spots at the site of sample application (strip b). These spots were found to be the result of precipitation of calcium salts in blood plasma b y the a4dition of relatively low specific activity Ca 45. When radiocalcium of higher specific activity was added to serum (strip c) all the radioactivity was found in the calcium-ion band. These results show that the bond between calcium and serum protein is so weak that it is easily and r a p i d l y broken by the electrophoretic removal of free calcium ions f r o m the medium s u r r o u n d i n g the protein molecules. To compare the physico-chemical state of calcium in milk to that in blood, ultrafiltration and p a p e r electrophoresis were p e r f o r m e d on a milk sample incubated with Ca4'~Cle for several hours. The electrophoretic p a t t e r n lacked definition. Radioactivity was distributed more or less evenly between the origin and an ionic-calcium b a n d on the cathode side, and radioactivity was also found with the protein bands toward the anode. Such a picture could be explained by the presence of protein-bound or other colloidal calcium, which decomposed
SECRETION
OF
C a *~ B Y
THE
MAMMARY
GLANDS
1599
FI~. 6. A u t o r a d i o g r a m s of Ca4:CI~ (a) a n d Ca~C12 in blood s e r u m (b a n d e) s u p e r i m p o s e d on p a p e r eleetrophoretie s t r i p s s t a i n e d to show the s e r u m p r o t e i n s n e a r 0. T h e lower b a n d s a r e t h e i m a g e s of Ca% S a m p l e w a s a p p l i e d a t 0 - > .
slowly u n d e r the influence of electrophoretic w i t h d r a w a l of Ca ions f r o m the medium. Ultrafiltration of the same milk sample over a period of 16 hours in the r e f r i g e r a t o r indicated the presence of 72% nondiffusible calcium, a value closely agreeing with the results of Magee and H a r v e y (10). Ca 45 determinations on the ultrafiltrate showed t h a t its specific activity exceeded the specific activity of whole milk b y 33%. A p p a r e n t l y the calcium in milk did not exchange freely with ionic calcium as it had with serum calcium.
Effect on blood and milk calcium of intravenous injection of large amounts of stable calcium. Although all methods of blood fractionation used h a d failed to show a high specific activity component, the a p p e a r a n c e of the excess milk calcium specific activity a f t e r oral doses suggested t h a t recently absorbed calcium might be used for milk f o r m a t i o n in slight preference over the total blood calcium. I f this were true, it was reasoned t h a t adding extra calcium to the blood to sinmlate newly absorbed calcium should produce a g r e a t e r effect upon the specific activity of milk calcium t h a n upon the blood of a cow in the straight declining phase a f t e r an oral dose. To test this hypothesis, a cow t h a t h a d previously shown the a p p a r e n t Ca 4~ concentrating effect in milk was used in
1600
E.
V',;.
SWANSON ET AL
three different trials. I n each trial, the cow was given a 5-mc. oral dose of Ca~C12, and blood and milk samples were collected as previously described. Beginning at about 30 hours after dosing, injections of solutions furnishing 0.5 g. of Ca were made by intravenous catheter at 30-minute intervals for 8 hours. D u r i n g this injection schedule, milkings were made at 3- to 4-hour intervals. Blood samples were taken just before an injection at 2-hour intervals or oftener. I n the first trial, CaCl2 solution was used. I n the second trial, CaCle and NaI~2P04 solutions were used in proper proportion to maintain a normal C a : i ~ ratio in the blood. Calcium lactate was used in the t h i r d trial. The results of the preliminary oral dose with cow 8 and the injection of calcium plus phosphorus trial with the same cow are shown in Figures 7 and 8.
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FI~. 7. Comparison of the specific activity of blood and milk calcium of cow 8 after an oral dose of Ca4G given near the peak of her lactation, December 10, 1952.
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~'m. 8. Changes in the specific activity of blood and milk calcium after an oral dose of Ca~; given to cow 8 March 4, 1953, followed by 8 hours of injecting CaCl.~ and NaII._.PO, March 5.
I t is shown in these graphs that a much greater difference between milk anent blood calcium specific activity was f o u n d in the preliminary trial than in the later ones. I n the former trial the cow was at the peak of her lactation, 5 weeks, in December, 1952. The CaC12 trial was made in J a n u a r y , 1953, and the next trial in March, 1953. I n all trials the specific activity of calcium from blood a n d milk declined at the same rate, producing parallel curves. The results with CaC12 or calcium lactate were very similar to those with calcium and phosphorus. C o n t r a r y to the above hypothesis, the specific activity of milk was influenced less by massive injections of stable calcium than was that of blood. The plotted differences are probably less than the true average differences because the blood was sampled at its highest specific activity within each 30-minute injection period. The CaC12 injections decreased blood calcium specific activity 19%, but at the same time milk calcium specific activity decreased only 11%. Calcium and phosphorus injected together caused a 12% decrease in blood and
1601
S E C R E T I O N OF Ca ~n BY T H E MAMMARY G L A N D S
an 8% decrease in milk calcium specific activity. In neither trial did the altered specific activity of milk calcium decline to the normal expected blood level. Although the distance between the milk and blood specific activity curves indicated a probable average lag of 7 and 9 hours, respectively, in the two trials, the lag in appearance of the effects of the calcium injections on nfilk calcium specific activity was less than 4 hours. This indicates that p a r t of the milk calcium is derived rapidly from the blood while another p a r t may pass through an udder reservoir, which would cause a delay in the movement from blood to nfilk. The contribution of this u d d e r calcium reservoir to the milk explains why the specific activity of milk calcium was affected less than blood calcium by the 8 hours of calcium injections. These results indicate that injected calcium is not preferentially used for milk formation to the extent that it may explain the peak differences in blood and milk calcium specific activities. Isotopic analysis of blood a.nd milk calcium. I f the cow's m a m m a r y gland were capable of preferentially selecting Ca 45 from the blood for secretion into milk, the differences in milk and blood specific activities might be due simply to such an isotope effect. I f this were true, milk should also be richer than blood in stable calcium isotopes Ca 4~ and Ca ~. To determine this, blood and milk samples were secured from a cow in early lactation. Caleiunl was collected from each as the oxalate precipitate and then converted to the nitrate and analyzed by the mass spectrograph. The percentages of six stable calcium isotopes found in blood and milk samples are shown in Table 1. Since the TABLE
J
The eo~ce~tratio~ of six stable ealci~¢m isotopes it~ blood a~d ~ill~ draw~ f*'om the sa:me cow Concentrstion Calcium isotope
Blood
in atom per cent Milk
(~ass No.)
(%)
(%)
40 42 43 44 46 48
96.81 0.68 0.14 2.]8 0.003 0.189
96.81 0.66 0.15 2.19 0.003 0.193
values of these six isotopes were nearly identical from the two sources of calcium, it seems unlikely that the m a m m a r y gland of the cow concentrates Ca ~'~ per se; thus, the higher specific activity of nfilk may not be explained by an i s o t o p e effect. ComparisoJ~ of specific activity changes of calcium ,in arterial a~d venous blood, urine, a~d milk of cows after a~ oral dose of Ca '~. In all of the previous trials blood samples had been obtained from the jugular vein. Since blood calcium is constantly being exchanged with bone calcium, the blood is constantly being depleted of recently absorbed calcium (in this instance Ca 45) which is replaced by calcium (mostly stable Ca) from the skeletal and tissue stores (7, 14). Theoretically, this could result in venous calcium having a v e r y slightly
1602
E.w. swA~sos ET A n
lower specific activity than arterial calcium. Another possible change in blood calcium could occur as the result of the removal of calcium by the udder. An attempt was made to detect such changes in this experiment. Three cows were used in these trials. About a week before dosing, the carotid a r t e r y of each cow was displaced so that it lay just under a fold of the skin of the neck. Arterial blood samples were then easily obtained by hypodermic needle and syringe. Each cow was dosed with 5 me. of Ca~sCl=, orally. Blood samples were taken at frequent intervals sinmltaneously from the carotid artery, j u g u l a r vein, and subcutaneous abdominal vein, which was presumed to contain mostly blood from the m a m m a r y gland. Milk and urine samples were obtained twice daily. Because of its v e r y low calcium content, urine was wet-ashed in glass beakers to insure reliable specific activity values. The specific activity values of calcium from each source from cows 7 and 9 are presented in Figures 9 and 10. Similar results were secured from cow 8. • MAM.VEIN X JUG.VEIN • ARTERY
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FI(~. 9. C o m p a r i s o n s of t h e specific activi t y of calcium f r o m milk ( b r o k e n line) a n d u r i n e a n d blood f r o m t h e c a r o t i d a r t e r y , j u g u l a r vein, a n d s u b c u t a n e o u s a b d o m i n a l vein (solid line) of cow 7 a f t e r a n oral dose of Ca 4G.
FIG. 10. C o m p a r i s o n s of t h e specific activity of c a l c i u m f r o m milk ( b r o k e n line) a n d u r i n e a n d blood f r o m t h e carotid •~rtery, j u g u l a r vein, a n d s u b c u t a n e o u s abd o m i n a l vein (solid line) of cow 9 a f t e r a n oral dose o f Ca 'G.
No consistent difference in specific activities o f calcium from urine, arterial blood, jugular vein blood, or subcutaneous abdominal ( m a m m a r y ) vein blood was found. Therefore, the t r e n d of these values has been represented by one line. The specific activity of milk calcium from these cows did not reach a higher level than that of blood except in the declining phase of the curves, at which time the specific activity of calcium from the three blood sources continued to remain nearly identical. I f slight differences in blood specific activity did exist, the analytical methods were not precise enough to demonstrate them. The agreement of specific activity of urine and blood calcium in nearly all samples indicates that the calcium excreted by the kidney is representative of all of the blood calcium, lending f u r t h e r doubt as to the existence of a high specific activity blood fraction. Since only one of the cows (No. 9 in F i g u r e 10) used in this experiment demonstrated the differences in milk and blood calcium specific activity expected on the basis of previous trials, these results may not have been typical of cows
SECRETION
O F C a 4~ B Y
TItE
1603
I~IAMMARY GLANDS
in which this effect did occur. However, cows 7 and 8 had exhibited definite differences in milk and blood calcium specific activities in previous experiments. The specific activities of blood calcium f r o m the three sources differed no more for cow 9 t h a n for the two cows showing lesser blood-milk differences. Two other trials comparing the specific activities of serum calcium f r o m the j u g u l a r and subcutaneous abdominal veins also failed to show differences large enough or consistent enough to explain the blood and milk differences. Other observations on the secretion of Ca ~ into milk. Cow 8, which h a d a history of milk fever in previous lactations, r e p e a t e d l y showed a higher p e a k specific activity in milk t h a n in blood calcium, a n d the differences between blood and milk calcium specific activities were wider in early lactation ( F i g u r e 7) t h a n in later lactation ( F i g u r e 8). Another cow (No. 87) with a history of milk fever was also used in Ca 4~ secretion studies a n d was given oral doses in the first and f o u r t h months of lactation. The results f r o m these trials are shown in F i g u r e 11. As with cow 8, wide differences in milk and blood calcium 0
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FIG. 12. Comparison of the specific act i v i t y of blood a n d milk calcium f r o m cow 6 f o r 10 days a f t e r the last of 10 daily intravenous injections of Ca 4~, J u l y , 1952.
specific activities were observed at the peak of lactation, a n d practically no differences were found at the later trial. These results suggest t h a t the process by which the specific activities differences are produced is most active in early lactation. T h a t the stage of lactation is just one of the factors involved, however, is shown b y the results with cows 1 and 5 at two different periods (Figures 1 and 2) and b y the results with cow 9 ( F i g u r e 10) and cow 11 ( F i g u r e 5), which were both good producers used in the plotted experiments at an early
1604
~. w. SWANSONET AL 513 40
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TIME AFTER L A S T DOSE ( h r )
FIS. 13. Comparison of the specific activity of blood and milk (.}fl(.ium f r o m cow 7 for 10 days s f t e r the last of I0 daily i n t r a v e n o u s injections of Ca ~', July, 1952.
stage of lactation. I n these four cows only slight or no differences in peak specific activities of blood and milk calcium were observed from experiments in the early part of lactation. A n interesting comparison of the specific activities of blood and milk calcium was made with two cows that were used in an experiment to determine endogenous fecal calcium (16). These cows were given daily intravenous injections of 150 and 200 ~c. of Ca4'~Cl_~ for 10 days respectively, in each of two separate trials. The specific activities of milk and blood calcium for 10 days after the last injection in one trial are shown in Figures ]2 and 13. The values for blood and milk for cow 6 were nearly identical in both trials. F o r cow 7, however, the curves for blood and milk although parallel were separated by about 72 hours in time in both trials. These results indicate again that the decline in specific activity of blood and milk calcium is generally at the same r a t e ; therefore, the udder probably does not concentrate Ca 4~ continuously. The consistent difference in the response of the two cows indicates again the wide variations that lnay exist in the manner in which calcium is handled by the cow or the udder. The milk production of cow 6 ia both trials was 8 to 10 lb. daily, whereas cow 7 was producing 15 to 18 lb. daily, and this production difference m a y have had an influence on the calcilun metabolism. DISC USSION
This series of experiments has helped to clarify some of the questions reg a r d i n g calcium metabolism by the milking cow, but the explanation for others is still obscure. No entirely satisfactory explanation of the higher specific ac-
SECRETION
O F Ca 4~ B Y T H E
MAMMARY
GLANDS
1605
tivity of milk calcium than blood calcium at the peak of the curves after an oral dose has appeared. This phenomenon has also been reported for phosphorus after an oral dose of p32 (13) with the supposition that the m a m m a r y gland removed from the blood a high specific activity fraction of phosphorus for milk secretion. All attempts, including those reported here and work by Visek et al. (15), to demonstrate such a high specific activity fraction of calcium have resulted in negative observations. I f such a blood fraction exists it must be handled differently in different cows, since not all cows have consistently shown the higher peak milk specific activity. This is not a remote possibility, however, since cows are known to differ considerably in calcium metabolism and blood calcium components just after parturition, resulting in differences in milk fever development; and, furthermore, cows in these experiments known to be susceptible to milk fever were among the most definite demonstrators of the differences in blood and milk calcium specific activity. It is postulated that if such a high specific activity blood calcium fraction does exist, its duration is relatively short after oral dosing. Otherwise, as time progressed the specific activities of blood and milk calcium would diverge markedly r a t h e r than remain parallel, as they have in nearly all of these studies. Additional evidence against a high specific activity fraction of blood calcium is the observation that calcium in the urine and that in blood were n e a r l y identical in specific activity. These results confirm similar observations in dogs (5) and humans (1). If blood calcium fractions of differing specific activities were circulating, it would be unlikely that the kidney would excrete them in the same proportion as that f o u n d in blood. F u r t h e r m o r e , lack of specific activity differences between arterial blood calcium and m a m m a r y vein blood calcium did not indicate that the m a m m a r y gland was removing a high specific activity fraction. Since the total calcium arterial-venous differences in this study were only about 2 to 4% and the peak specific activity differences wl~en exhib.ited were usually of the order of 10 to 15%, the precision of the analytical methods may not have been good enough to show such an effect. Failure to alter milk calcium specific activity as much as that of blood calcium by repeated intravenous doses of calcium suggested that blood calcium recently absorbed largely as ionic calcium may not be preferentially used for milk formation, supposing that absorbed and injected calcium would react similarly. The mass spectrographic analyses of blood and milk calcium from one cow gave no indication of the possibility of a preferential secretion by the mamm a r y gland of heavier than average calcium isotopes. I n an experiment subsequent to the samplings for spectrographic analysis, it was found that this cow (No. 10) did not appear to concentrate Ca 45 in her milk after an oral dose. Until the isotopic analyses can be made on blood and milk of a cow showing the concentrating effect, the possibility of isotope separation in some cows should not be completely rejected. I f there were an isotope separation, however, it would not be expected that the heavy isotopes would enter into the reactions leading to milk formation at a greater rate than the light isotopes. Therefore, this explanation is given v e r y minor consideration.
1606
E.W. SWANSON ET AL
A f t e r the peaks of activity have passed, the specific activity of milk calcium corrected to the average time of secretion of the sample has in a great m a j o r i t y of the experiments remained above the specific activity of blood calcium as long as reliable samples could be collected. The rate of decline of both blood and milk values has been exponential, and the values have been parallel. This situation is consistent with the hypothesis t h a t milk calcium is derived f r o m a homogeneous portion of blood calcium but t h a t a constant delay in the appearance of the milk calcium occurs. This delay can be explained by the existence in the u d d e r of a calcium reservoir f r o m which a large p a r t of the milk calcium is eventually drawn. Such a calcium pool has been suggested by Gowen and Tobey (6) on the basis of excessive ash content of the udder. Monroe (11) found that four udders f r o m cows in late lactation contained two to four times as m u c h calcium as could be accounted for b y the residual milk they contained. I t is possible that udders f r o m cows at the peak of lactation would show even greater stores of excess calcium. Since the total residual milk m a y easily equal 20% of the amount n o r m a l l y secured at a milking, it follows that the recently milked u d d e r m a y contain enough calcium to f o r m the amount of milk to be secreted in the next 12 hours. Another i m p o r t a n t factor in the movement of calcium f r o m blood through the u d d e r to milk is the relatively low exchangeability of milk calcium, which has been demonstrated in these experiments and others (17). I t is postulated t h a t calcium which enters the secretory tissue of the u d d e r is also bound in some m a n n e r so that it does not again r e t u r n easily to the blood but is p r i m a r i l y released or continued in processing to f o r m milk. Such a binding process is necessary to explain the v e r y long periods that have been observed in some cows between equal specific activities of blood and milk calcium. One cow dosed p r e p a r t u m (15) had shown such a lag period of about 10 days for milk secreted d u r i n g the first 10 days of lactation. Several cows in the experiments reported here have shown lag periods of 18 hours or more, and one period was 72 hours. Such long delays cannot be explained by the delay of movement of p r e f o r m e d milk through the udder, as was proposed by L a k s h m a n a n and K u m a r (9). On p u r e l y physical considerations such m~ explanation is not plausible because the total weight of a milked udder is not equal to more than one d a y ' s milk yield. The variability in the time lag between equal calcium specific activities of Mood and milk is p r o b a b l y due to a eombination of m a n y fat'tors. The size of the u d d e r calcium reservoir and its proportionate eontribution to milk calcium is one factor. The data in F i g u r e 8 show that p a r t of the milk ealeimn is derived r a p i d l y f r o m the blood, probabl.v without passing th,'ough the udder reservoir. I f all of the nlilk calcium came d i r e , t l y f r o m blood as tim milk was secreted, the specific activity curves of blood and milk would be expected to coincide in the declining phase, as they have in several of the experiments presented here. I f the calcium binding reservoir of the udder were very large or active, proportionately more of the milk would be derived from this source, which would result in longer lag periods. Another possibility is a variation in exchange-
SECI~ETION OF Ca4~ BY THE MAMMARY GLANDS
1607
ability of udder calcium. The differences observed could be explained without assuming m a j o r changes in the amount of calcium reserve in the u d d e r if exchange rates varied widely. E x t r e m e l y r a p i d exchange would result in a minim u m time lag, whereas a low exchange rate with a given size reservoir would produce a longer period between equality of blood and milk calcium specific activity. More definitive experiments u p o n secreting m a m m a r y tissue are necessary to clarify these questions. SUMMARY
E x p e r i m e n t s have been discussed in which the specific activities of milk calcium and blood calcium have been compared a f t e r intravenous and oral doses of radioactive calcimn administered to lactating d a i r y cows, along with investigations to determine the cause of the differences observed. A f t e r intravenous doses the specific activity curve of blood calcium as it declined passed t h r o u g h the peak of the milk calcium curve corrected to average time of secretion. A f t e r the peak, the milk specific activity usually remained higher t h a n the blood but continued to decline at the same rate as blood. A f t e r oral doses wide variations in response have been observed. I n some animals the specific activity of milk calcium has exceeded the highest observed specific activity of blood, but in others they were nearly equal at their maxinmm. A f t e r the peak the decline curves have been v e r y similar to those a f t e r an intravenous dose. Cows that had previously developed milk fever were f o u n d to exhibit greater differences ill specific activities of blood aud milk calcium in early lactation t h a n other cows; yet these differences were observed also in cows t h a t were not highly susceptible to milk fever. No satisfactory explanation for the excessive p r o p o r t i o n of radioactive calcium in milk has been discovered. P a p e r electrophoresis, arterial-venous differences, and experiments involving the constant injection of stable calcium failed to s u p p o r t the hypothesis t h a t a high specific activity fraction of blood calcium existed which m i g h t be used p r e f e r e n t i a l l y for milk formation. Mass spectrographic analysis of blood a n d milk calcium of one cow showed no differences in proportions of six calcimn isotopes, which indicated that the u d d e r was not concentrating Ca 45 per se. The existence in the u d d e r of a variable calcium reservoir f r o m which the calcium is not readily exchanged with blood calcium, and which is used for milk formation, has been postulated to explain the differences in time between equal specific activity values of blood and milk calcium. ACKNOWLEDGMENTS T h e t e c h n i c a l a s s i s t a n c e of I r a W h i t n e y , G. W. R o y s t e r , a n d T. G. Clark in m o s t of t h e s e e x p e r i m e n t s is g r a t e f u l l y acknowledged. T h e n m s s s p e c t r o g r a p h i c a n a l y s e s of blood a n d milk c a l c i u m were done b y Oak R i d g e N a t i o n a l L a b o r a t o r y t h r o u g h t h e c o u r t e s y of R. F . t t i b b s . REFERENCES ( ] ) BLAU, M., tion of (2) COMAR, C. U s e of
SPENCER, H., S~VERNOV, J-., AND LASZLO, ]). U t i l i z a t i o n a n d I n t e s t i n a l ExcreC a l c i u m in M a n . Science, 120: 1029. 1954. L., HANSARD, S. L., HOOD, S. L., I:)LUMLEE, M. P., AND BARRENTINE, B. F . Calcium-45 in Biological Studies. N~lcleonics, 8: 19. 1951.
1608
E. W. S W A N S O N E T A L
(3) ELLENBEI~GER, H. B., NEWLANDER, J. A., AND JONES, C. H. Calcium and Phosphorus Requirements of Dairy Cows. II. Weekly Balances Through Lactation and Gestation Periods. Vt. Agr. Expt. Sts., Bull. 342. 1932. (4) GOr~DON, A. H., GROSS, J., O'CoNNoR, D., AND PITT-RIVERS, R. Nature of the Circulating Thyroid Hormone-Plasma Protein Complex. Nat~lre, 169: 19. 1952. (5) GOVAERTS, J. Studies in Calcium Urinary Excretion with the Aid of Radiocalcium. A ~ . J. Physiol., 159: 542. 1949. (6) GOWEN, J. W., AND TOB]~Y, E. R. Significance of Chemical Composition of the Secreting and Dry Mammary Gland to Milk Secretion. J. Gen. Physiol., 12: 123. 1928. (7) HANSARD, S. L., COMAR, C. L., AND DAVIS, G. K. Effects of Age Upon the Physiological Behavior of Calcium in C a t t l e . . 4 ~ . J. Physiol., 177: 383. 1954. (8) HIBBS, J. W. Milk Fever (Parturient Paresis) in Dairy Cows--A Review. J. Dairy Svi., 33: 758. 1950. (9) LAI,ISCHMANAN, S., AND KUAIAI¢, S. Specific Activity-Time Relationship in Tracer Studies of Milk Secretion. J. Dairy Sci., 37: 670. ]954. (10) MAGEE, H. E., AND H_~KVEY, D. Studies on the Effect of Heat on Milk. I. Some Physiochemical Changes Induced in Milk by Heat. Biochen~. J., 20: 873. 1926. (11) M0~R0s, R. A. Unpublished data from the U.T.-A.E.C. Agricultur:d Research Program. (12) NIEDE~.M]~IEK, R. P., SMITH, V. R., AND WHITKHAIR, C. K. Parturient Paresis. I I I . A Study of Various Blood Constituents at Parturition in Masteetomized Cows. J. Dairy Sci., 32: 927. 1949. (13) SAARINE2q, P., C05IAR, C. L., MARSHALL, S. P., AND DAVIS, G. K. Partition of Orally Administered Radioactive Phosphorus in the Blood and Milk of the Dairy Cow. J. Dairy 8ci., 33: 878. 1950. (14) TtIOMAS, R. O., LITOVITZ, T. A., RUBIN, M. or., AND GESCHH,:TE[¢, C. F. Dynamics of Calcium Metabolism. Time Distribution of Intravenously Administered Radioealeium. Am. J. Physiol., 169: 568. 1952. (15) VISEK, W. J., MONROE, R. A., SWANSON, E. W., AND CO.~X.XR,C. L. Calcium Metabolism in Dairy Cows as Studied with Ca *'~. J. Dairy 8ci., 36: 373. 1953. (16) VISF~K, W. J., MONROZ, R. A., SWANSON, E. W., AND COMAU, C. L. Determination of Endogenous Fecal Calcium in Cattle by a Simple Isotope Dilution Method. J. Nutrition, 50: 23. 1953. ( ] 7 ) WRIGHT, N. C. The Mechanism of Secretion of Calcium "tad Phosphorus in Milk. J. Agr. Sci., 18: 478. 1928. (18) ZILVERSMIT, D. B., ENTENMAIk', C., AND FISItLER, M. C. On the Calculation of " T u r n over T i m e " and " Turnover R a t e " from Experiments Involving the Use of Labeling Agents. J. Gem Pl, ysiol., 25: 325. 1943.
OF VARIATIONS
IN SECRETION
OF
Ca ~5 B Y T H E
M A M M A R Y G L A N D S O F D A I R Y C O W S 1,2 E. W. SWANSON, R. A. MONROE, 3 D. B. ZILVERSMIT,~ W. J. VISEK, 5 AND C. L. COMAR~
Depart~aent of Dairying, University of Tennessee, Knoxville, and Ato,nic Energy Commission Agric,~dtural Research Program, Oat~ Ridge, Tennessee
A better understanding of the causes of low blood calcium after parturition is important for proper control of milk fever. This series of experiments has helped clarify some of the questions regarding calcium metabolism, though no satisfactory explanation for the excessive proportion of radioactive calcium in milk has been found. E d i t o r .
The c a l c i u m m e t a b o l i s m of the l a c t a t i n g d a i r y cow proceeds at a h i g h r a t e because of the secretion into m i l k of 20 to 30 g. of c a l c i u m each day. This is a b o u t f o u r times the c a l c u l a t e d fecal metabolic loss of c a l c i u m b y the cow (16). B a l a n c e s t u d i e s have shown t h a t the l a c t a t i n g cow is n o r m a l l y i n n e g a t i v e c a l c i u m b a l a n c e i n the m a j o r p a r t of the l a c t a t i o n period, even w h e n a d e q u a t e l y fed (3). The f u n c t i o n i n g u d d e r has been s h o w n to be the cause of drastic c h a n g e s i n c a l c i u m c o n t e n t of the blood at the i n i t i a t i o n of l a c t a t i o n (12), a n d d a i r y cows are u n i q u e l y afflicted with a n a i l m e n t , m i l k fever, caused b y low blood c a l c i u m a f t e r p a r t u r i t i o n (8). A n u n d e r s t a n d i n g of the secretion of milk c a l c i u m a n d its p r o b a b l e p r e c u r s o r forms a n d sources has b e e n s o u g h t i n previous s t u d i e s b y the use of r a d i o a c t i v e c a l c i u m b y which blood c a l c i u m a n d feed c a l c i u m have been l a b e l e d (15). These s t u d i e s i n d i c a t e d t h a t some cows a p p a r e n t l y were able to c o n c e n t r a t e r a d i o a c t i v e c a l c i u m i n t h e i r m i l k a f t e r oral d o s i n g to a g r e a t e r e x t e n t t h a n t h e y c o n c e n t r a t e d stable calcium, a l t h o u g h this p h e n o m e n o n was n o t so n o t i c e a b l e a f t e r i n t r a v e n o u s dosing. A f t e r both oral a n d i n t r a v e n o u s doses, the p r o p o r t i o n of r a d i o a c t i v e to stable c a l c i u m r e m a i n e d h i g h e r in milk t h a n i n blood, even a f t e r c o r r e c t i o n for time of m i l k secretion. Si,we this difference in the p e a k specific a c t i v i t y of blood a n d m i l k c a l c i u m was Received for publication March 30, 1956. 1 This manuscript is published with the permission of tile Director of the University of "remlessee Agricultural Experiment Station, Knoxville. 2 This work was completed under Contract No. AT-40-1-GEN-242 between the University of Tennessee, College of Agriculture, and the Atomic Energy Commission. The radioactive materials used in this work were obtained from the Oak Ridge National Laboratory on allocation from the United States Atomic Energy Commission. Present address: University of California, Atomic Energy Project, West Los Angeles. 4 Present address: Department of Physiology, University of Tennessee, l~Iemphis. Present address: University of Chicago, Medical School. " Present address: Oak Ridge Institute of Nuclear Studies, Medical Division, Oak Ridge. 1594
SECRETION
OF Ca 4n B Y T H E
51AMMARY G L A N D S
1595
at variance with the expected precursor-product relationship (18), the additional studies presented in this article were coudueted to seek a f u r t h e r explanation for this phenomenon. MATERIALS
AND
METHODS
The cows used in this study either were all Jerseys or carried at least 50% J e r s e y blood and were mature in age. They varied widely in milking ability and stage of lactation through the series of experiments. They were fed a medium to poor quality mixed hay and commercial 16% protein dairy feed which was fortified with mineral supplements containing adequate calcimn, phosphorus, and minor elements, l)osing, sampling, and analyses were done by the methods previously described (2, 15). Intravenous injection of 10 units oxytocin was given prior to all milkings during the first 2 days of each experiment and for all later periods in which frequent milkings were made. Special methods for individual experiments are described below with the discussion of those experiments. Specific activity of calcium has been calculated as the percentage of the dose of Ca ~5 times 10 * per milligram of total calcium. In plotting the specific activities of calcium from milk and urine, the median time between sample collections has been used r a t h e r than the time of collection. This correction for time of secretion of milk was assumed to be adequate because it was f o u n d that the specific activities of first and last drawn milk a f t e r oxytocininduced milk ejection at one milking period differed only slightly, being 1.93 and 1.83, respectively. All changes in specific activity with time have been plotted on semilogarithmic scales, according to time after dosing. RESULTS
Comparing oral and intravenous doses. Since comparisons of specific activity of blood and milk calcium after oral and intravenous doses in the same cow had not been presented previously, these data were obtained from two cows in mid-lactation. They were given oral doses of approximately 5 inc. Ca45C12 December 14, 1951, and F e b r u a r y 1, 1952. They were dosed intravenously with 1.75 inc. of Ca45C12 ~[arch 5, 1952. The results of these trials with cow 1 are shown in Figures I to 3. V e r y similar results were obtained in the trials with cow 5. These figures show that the declining curve of blood calcium specific activity a f t e r an intravenous dose passed through the peak of the time-corrected milk curve, but later a slight delay in calcium secretion into milk was indicated. In the straight declining phase of the curves the blood and milk values were parallel b.~lt separated by a time factor of about 8 hours. These differences might be explained by a calcium reservoir in the udder requiring an average of 8 hours for milk precursor calcium to traverse. The specific activity curves a f t e r oral doses were somewhat different. In the December trials the peaks of milk and blood calcium specific activity were nearly equal, but in the F e b r u a r y £rials milk reached a higher specific activity than blood, as had been noted
1596
E. ~V. SWANSON- ET AL
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]~m. 1. C o m p a r i s o n of the specific act i v i t y of blood a n d m i l k c a l c i u m a f t e r an o r a l dose of Ca ~ g i v e n to cow I D e c e m b e r 14, 1951.
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Fro. 3. C o m p a r i s o n of the specific act i v i t y of blood a n d m i l k c a l c i u m a f t e r all i n t r a v e n o u s dose of Ca ~ g i v e n to cow 1 M a r c h 5, 1952.
'
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'
'
40
'
'
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'
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'
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FIG. 4. C o n l p a r i s o n of t he specific act i v i t y of blood a n d m i l k c a l c i u m a f t e r a n oral dose of Ca 45 g i v e n to cow 5 w i t h collec t i ons of s a m p l e s a t 4 to 6 h m l r i n t e r v a l s .
before. The straight declining blood and milk curves for cow 1 were nearly parallel, but for cow 5 they seemed to diverge in later samplings. The time differences between the curves for cow I were 13 and 17 hours and those for cow 5 were 14 to 20 hours and 20 to 26 hours, respectively, in the two trials. I f the u d d e r were p r e f e r e n t i a l l y concentrating radioactive calcium, which after an oral dose is a representative tracer of feed calcium, the specific activity decline curves of blood and milk would be expected to diverge. The observation
SECRETION OF Ca~ BY TItE MAMMARYGLANDS
1597
that they diverged with one cow and not with the other, irrespective of tile relation of milk and blood calcium specific activities at the peaks, makes the i n t e r p r e t a t i o n of this phenomenon difficult. These types of inconsistencies have a p p e a r e d at other times in these studies. There was a strong suggestion, however, t h a t the cow's m a m m a r y gland m a y handle orally derived calcium differe n t l y f r o m t h a t placed into the blood stream all at one time. Blood and milk calcium specific activity changes after oral doses. The possibility of the blood calcium reaching a higher peak specific activity t h a n t h a t of milk a f t e r an oral dose, but m a i n t a i n i n g it for so short a time that it was missed in the sampling schedule, was investigated with two cows f r o m which v e r y frequent samples were taken in the early phase of the trial. E a c h cow was dosed orally with about 5 inc. of Ca4~Cl~o. I f the contents of the dosing capsule were not thoroughly mixed in the cow's stonlach but were r a p i d l y shunted to an area where the m a j o r portion was absorbed, such a t r a n s i t o r y peak blood specific activity would be possible. 5.0
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]~m. 5. Comparison of the specific gravity of blood and milk calcium after an oral dose of Ca~ given to cow 11 with frequent collection of samples. The results of these trials are shown in F i g u r e s 4 and 5. E v e n with samples t a k e n at 4-hour intervals, the specific activity of none of the blood samples f r o m cow 5 was as high as t h a t of the seven highest milk samples ( F i g u r e 4). The peak specific activity of the blood was not attained until 34 hours a f t e r dosing, which indicates a r a t h e r slow absorption of the dose. The peak specific activity a p p e a r e d in the milk at about the same time as the blood peak. I n cow 11, however, peak specific activity was f o u n d in the 12-hour blood samples, which exceeded the highest specific activity f r o m a n y of the milk samples (Figure 5). The peak for specific activity in milk occurred at about 20 hours. These
1598
E.w.
SW.(NSON ET .(L
two cows clearly absorbed and metabolized their ingested calcium at different rates. Cow 11, which absorbed the dose more rapidly, failed to concentrate the radioactive calcium in the milk as did cow 5, which absorbed the dose more slowly. The specific activity curves of the blood and milk of both cows appeared to diverge slightly in the declining or p r e s u m a b l y post-absorptive phase. These first experiments seemed to confirm the previous observation (15) that the mamm a r y gland was concentrating radioactive calcium in preference to stable calcium a f t e r an oral dose in some cows at some periods; but the effect was not always observed.
Test for a high specific activity blood fraction by electrophoresis after adding Ca ''~. Although Visek et al. (15) had not been able to find blood fractions that differed significantly in specific activity f r o m the whole blood calcium by means of ultrafiltration and adsorption, the possibility t h a t serum calcium is not all of equal mobility still exists. One example which m i g h t be due to differences in mobility of radioactive calcium in the bloocl is the observation that specific activity of blood calcium of the new-born calf f r o m a cow dosed before p a r t u r i tion was 2.5 times t h a t of the cow (15). A f u r t h e r a t t e m p t to detect differences iu blood calcium fractions was made by a combination of p a p e r electrophoresis and autoradiographic techniques. The method of Gordon et al. (4) was used. All strips were moistened with 0.05 M veronal buffer ( p H 8.5) and 5 to 10 )t of sample were applied to the moist strip before a potential gradient of 5 volts per centimeter was established. The results of three typical tests arc shown in F i g u r e 6. Strip a is an autor a d i o g r a m of a sample of Ca4"~C12 a f t e r 1 hour electrophoresis in veronal containing 0.9% NaC1. All of the radioactive calcium had m i g r a t e d a distance of 7 cm. Strips b and c resulted f r o m the same t r e a t m e n t of bovine serum, which had been incubated with Ca 4n. The a u t o r a d i o g r a m is superimposed upon the p a p e r strip, which was stained with bromphenol blue to show the proteins. I t is evident t h a t the radiocalcium had m i g r a t e d the same distance as the calcium chloride (strip a) and t h a t it had separated cleanly f r o m the plasma proteins except for a few intensely radioactive spots at the site of sample application (strip b). These spots were found to be the result of precipitation of calcium salts in blood plasma b y the a4dition of relatively low specific activity Ca 45. When radiocalcium of higher specific activity was added to serum (strip c) all the radioactivity was found in the calcium-ion band. These results show that the bond between calcium and serum protein is so weak that it is easily and r a p i d l y broken by the electrophoretic removal of free calcium ions f r o m the medium s u r r o u n d i n g the protein molecules. To compare the physico-chemical state of calcium in milk to that in blood, ultrafiltration and p a p e r electrophoresis were p e r f o r m e d on a milk sample incubated with Ca4'~Cle for several hours. The electrophoretic p a t t e r n lacked definition. Radioactivity was distributed more or less evenly between the origin and an ionic-calcium b a n d on the cathode side, and radioactivity was also found with the protein bands toward the anode. Such a picture could be explained by the presence of protein-bound or other colloidal calcium, which decomposed
SECRETION
OF
C a *~ B Y
THE
MAMMARY
GLANDS
1599
FI~. 6. A u t o r a d i o g r a m s of Ca4:CI~ (a) a n d Ca~C12 in blood s e r u m (b a n d e) s u p e r i m p o s e d on p a p e r eleetrophoretie s t r i p s s t a i n e d to show the s e r u m p r o t e i n s n e a r 0. T h e lower b a n d s a r e t h e i m a g e s of Ca% S a m p l e w a s a p p l i e d a t 0 - > .
slowly u n d e r the influence of electrophoretic w i t h d r a w a l of Ca ions f r o m the medium. Ultrafiltration of the same milk sample over a period of 16 hours in the r e f r i g e r a t o r indicated the presence of 72% nondiffusible calcium, a value closely agreeing with the results of Magee and H a r v e y (10). Ca 45 determinations on the ultrafiltrate showed t h a t its specific activity exceeded the specific activity of whole milk b y 33%. A p p a r e n t l y the calcium in milk did not exchange freely with ionic calcium as it had with serum calcium.
Effect on blood and milk calcium of intravenous injection of large amounts of stable calcium. Although all methods of blood fractionation used h a d failed to show a high specific activity component, the a p p e a r a n c e of the excess milk calcium specific activity a f t e r oral doses suggested t h a t recently absorbed calcium might be used for milk f o r m a t i o n in slight preference over the total blood calcium. I f this were true, it was reasoned t h a t adding extra calcium to the blood to sinmlate newly absorbed calcium should produce a g r e a t e r effect upon the specific activity of milk calcium t h a n upon the blood of a cow in the straight declining phase a f t e r an oral dose. To test this hypothesis, a cow t h a t h a d previously shown the a p p a r e n t Ca 4~ concentrating effect in milk was used in
1600
E.
V',;.
SWANSON ET AL
three different trials. I n each trial, the cow was given a 5-mc. oral dose of Ca~C12, and blood and milk samples were collected as previously described. Beginning at about 30 hours after dosing, injections of solutions furnishing 0.5 g. of Ca were made by intravenous catheter at 30-minute intervals for 8 hours. D u r i n g this injection schedule, milkings were made at 3- to 4-hour intervals. Blood samples were taken just before an injection at 2-hour intervals or oftener. I n the first trial, CaCl2 solution was used. I n the second trial, CaCle and NaI~2P04 solutions were used in proper proportion to maintain a normal C a : i ~ ratio in the blood. Calcium lactate was used in the t h i r d trial. The results of the preliminary oral dose with cow 8 and the injection of calcium plus phosphorus trial with the same cow are shown in Figures 7 and 8.
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FI~. 7. Comparison of the specific activity of blood and milk calcium of cow 8 after an oral dose of Ca4G given near the peak of her lactation, December 10, 1952.
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~'m. 8. Changes in the specific activity of blood and milk calcium after an oral dose of Ca~; given to cow 8 March 4, 1953, followed by 8 hours of injecting CaCl.~ and NaII._.PO, March 5.
I t is shown in these graphs that a much greater difference between milk anent blood calcium specific activity was f o u n d in the preliminary trial than in the later ones. I n the former trial the cow was at the peak of her lactation, 5 weeks, in December, 1952. The CaC12 trial was made in J a n u a r y , 1953, and the next trial in March, 1953. I n all trials the specific activity of calcium from blood a n d milk declined at the same rate, producing parallel curves. The results with CaC12 or calcium lactate were very similar to those with calcium and phosphorus. C o n t r a r y to the above hypothesis, the specific activity of milk was influenced less by massive injections of stable calcium than was that of blood. The plotted differences are probably less than the true average differences because the blood was sampled at its highest specific activity within each 30-minute injection period. The CaC12 injections decreased blood calcium specific activity 19%, but at the same time milk calcium specific activity decreased only 11%. Calcium and phosphorus injected together caused a 12% decrease in blood and
1601
S E C R E T I O N OF Ca ~n BY T H E MAMMARY G L A N D S
an 8% decrease in milk calcium specific activity. In neither trial did the altered specific activity of milk calcium decline to the normal expected blood level. Although the distance between the milk and blood specific activity curves indicated a probable average lag of 7 and 9 hours, respectively, in the two trials, the lag in appearance of the effects of the calcium injections on nfilk calcium specific activity was less than 4 hours. This indicates that p a r t of the milk calcium is derived rapidly from the blood while another p a r t may pass through an udder reservoir, which would cause a delay in the movement from blood to nfilk. The contribution of this u d d e r calcium reservoir to the milk explains why the specific activity of milk calcium was affected less than blood calcium by the 8 hours of calcium injections. These results indicate that injected calcium is not preferentially used for milk formation to the extent that it may explain the peak differences in blood and milk calcium specific activities. Isotopic analysis of blood a.nd milk calcium. I f the cow's m a m m a r y gland were capable of preferentially selecting Ca 45 from the blood for secretion into milk, the differences in milk and blood specific activities might be due simply to such an isotope effect. I f this were true, milk should also be richer than blood in stable calcium isotopes Ca 4~ and Ca ~. To determine this, blood and milk samples were secured from a cow in early lactation. Caleiunl was collected from each as the oxalate precipitate and then converted to the nitrate and analyzed by the mass spectrograph. The percentages of six stable calcium isotopes found in blood and milk samples are shown in Table 1. Since the TABLE
J
The eo~ce~tratio~ of six stable ealci~¢m isotopes it~ blood a~d ~ill~ draw~ f*'om the sa:me cow Concentrstion Calcium isotope
Blood
in atom per cent Milk
(~ass No.)
(%)
(%)
40 42 43 44 46 48
96.81 0.68 0.14 2.]8 0.003 0.189
96.81 0.66 0.15 2.19 0.003 0.193
values of these six isotopes were nearly identical from the two sources of calcium, it seems unlikely that the m a m m a r y gland of the cow concentrates Ca ~'~ per se; thus, the higher specific activity of nfilk may not be explained by an i s o t o p e effect. ComparisoJ~ of specific activity changes of calcium ,in arterial a~d venous blood, urine, a~d milk of cows after a~ oral dose of Ca '~. In all of the previous trials blood samples had been obtained from the jugular vein. Since blood calcium is constantly being exchanged with bone calcium, the blood is constantly being depleted of recently absorbed calcium (in this instance Ca 45) which is replaced by calcium (mostly stable Ca) from the skeletal and tissue stores (7, 14). Theoretically, this could result in venous calcium having a v e r y slightly
1602
E.w. swA~sos ET A n
lower specific activity than arterial calcium. Another possible change in blood calcium could occur as the result of the removal of calcium by the udder. An attempt was made to detect such changes in this experiment. Three cows were used in these trials. About a week before dosing, the carotid a r t e r y of each cow was displaced so that it lay just under a fold of the skin of the neck. Arterial blood samples were then easily obtained by hypodermic needle and syringe. Each cow was dosed with 5 me. of Ca~sCl=, orally. Blood samples were taken at frequent intervals sinmltaneously from the carotid artery, j u g u l a r vein, and subcutaneous abdominal vein, which was presumed to contain mostly blood from the m a m m a r y gland. Milk and urine samples were obtained twice daily. Because of its v e r y low calcium content, urine was wet-ashed in glass beakers to insure reliable specific activity values. The specific activity values of calcium from each source from cows 7 and 9 are presented in Figures 9 and 10. Similar results were secured from cow 8. • MAM.VEIN X JUG.VEIN • ARTERY
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FI(~. 9. C o m p a r i s o n s of t h e specific activi t y of calcium f r o m milk ( b r o k e n line) a n d u r i n e a n d blood f r o m t h e c a r o t i d a r t e r y , j u g u l a r vein, a n d s u b c u t a n e o u s a b d o m i n a l vein (solid line) of cow 7 a f t e r a n oral dose of Ca 4G.
FIG. 10. C o m p a r i s o n s of t h e specific activity of c a l c i u m f r o m milk ( b r o k e n line) a n d u r i n e a n d blood f r o m t h e carotid •~rtery, j u g u l a r vein, a n d s u b c u t a n e o u s abd o m i n a l vein (solid line) of cow 9 a f t e r a n oral dose o f Ca 'G.
No consistent difference in specific activities o f calcium from urine, arterial blood, jugular vein blood, or subcutaneous abdominal ( m a m m a r y ) vein blood was found. Therefore, the t r e n d of these values has been represented by one line. The specific activity of milk calcium from these cows did not reach a higher level than that of blood except in the declining phase of the curves, at which time the specific activity of calcium from the three blood sources continued to remain nearly identical. I f slight differences in blood specific activity did exist, the analytical methods were not precise enough to demonstrate them. The agreement of specific activity of urine and blood calcium in nearly all samples indicates that the calcium excreted by the kidney is representative of all of the blood calcium, lending f u r t h e r doubt as to the existence of a high specific activity blood fraction. Since only one of the cows (No. 9 in F i g u r e 10) used in this experiment demonstrated the differences in milk and blood calcium specific activity expected on the basis of previous trials, these results may not have been typical of cows
SECRETION
O F C a 4~ B Y
TItE
1603
I~IAMMARY GLANDS
in which this effect did occur. However, cows 7 and 8 had exhibited definite differences in milk and blood calcium specific activities in previous experiments. The specific activities of blood calcium f r o m the three sources differed no more for cow 9 t h a n for the two cows showing lesser blood-milk differences. Two other trials comparing the specific activities of serum calcium f r o m the j u g u l a r and subcutaneous abdominal veins also failed to show differences large enough or consistent enough to explain the blood and milk differences. Other observations on the secretion of Ca ~ into milk. Cow 8, which h a d a history of milk fever in previous lactations, r e p e a t e d l y showed a higher p e a k specific activity in milk t h a n in blood calcium, a n d the differences between blood and milk calcium specific activities were wider in early lactation ( F i g u r e 7) t h a n in later lactation ( F i g u r e 8). Another cow (No. 87) with a history of milk fever was also used in Ca 4~ secretion studies a n d was given oral doses in the first and f o u r t h months of lactation. The results f r o m these trials are shown in F i g u r e 11. As with cow 8, wide differences in milk and blood calcium 0
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the specific
activity of blood and milk calcium of cow 87 a f t e r oral doses of Ca '5 J u l y 19, 1954 (olle m o n t h a f t e r calving with milk fever) a n d October 25, a f t e r milk yield h a d declined to a low level.
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FIG. 12. Comparison of the specific act i v i t y of blood a n d milk calcium f r o m cow 6 f o r 10 days a f t e r the last of 10 daily intravenous injections of Ca 4~, J u l y , 1952.
specific activities were observed at the peak of lactation, a n d practically no differences were found at the later trial. These results suggest t h a t the process by which the specific activities differences are produced is most active in early lactation. T h a t the stage of lactation is just one of the factors involved, however, is shown b y the results with cows 1 and 5 at two different periods (Figures 1 and 2) and b y the results with cow 9 ( F i g u r e 10) and cow 11 ( F i g u r e 5), which were both good producers used in the plotted experiments at an early
1604
~. w. SWANSONET AL 513 40
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TIME AFTER L A S T DOSE ( h r )
FIS. 13. Comparison of the specific activity of blood and milk (.}fl(.ium f r o m cow 7 for 10 days s f t e r the last of I0 daily i n t r a v e n o u s injections of Ca ~', July, 1952.
stage of lactation. I n these four cows only slight or no differences in peak specific activities of blood and milk calcium were observed from experiments in the early part of lactation. A n interesting comparison of the specific activities of blood and milk calcium was made with two cows that were used in an experiment to determine endogenous fecal calcium (16). These cows were given daily intravenous injections of 150 and 200 ~c. of Ca4'~Cl_~ for 10 days respectively, in each of two separate trials. The specific activities of milk and blood calcium for 10 days after the last injection in one trial are shown in Figures ]2 and 13. The values for blood and milk for cow 6 were nearly identical in both trials. F o r cow 7, however, the curves for blood and milk although parallel were separated by about 72 hours in time in both trials. These results indicate again that the decline in specific activity of blood and milk calcium is generally at the same r a t e ; therefore, the udder probably does not concentrate Ca 4~ continuously. The consistent difference in the response of the two cows indicates again the wide variations that lnay exist in the manner in which calcium is handled by the cow or the udder. The milk production of cow 6 ia both trials was 8 to 10 lb. daily, whereas cow 7 was producing 15 to 18 lb. daily, and this production difference m a y have had an influence on the calcilun metabolism. DISC USSION
This series of experiments has helped to clarify some of the questions reg a r d i n g calcium metabolism by the milking cow, but the explanation for others is still obscure. No entirely satisfactory explanation of the higher specific ac-
SECRETION
O F Ca 4~ B Y T H E
MAMMARY
GLANDS
1605
tivity of milk calcium than blood calcium at the peak of the curves after an oral dose has appeared. This phenomenon has also been reported for phosphorus after an oral dose of p32 (13) with the supposition that the m a m m a r y gland removed from the blood a high specific activity fraction of phosphorus for milk secretion. All attempts, including those reported here and work by Visek et al. (15), to demonstrate such a high specific activity fraction of calcium have resulted in negative observations. I f such a blood fraction exists it must be handled differently in different cows, since not all cows have consistently shown the higher peak milk specific activity. This is not a remote possibility, however, since cows are known to differ considerably in calcium metabolism and blood calcium components just after parturition, resulting in differences in milk fever development; and, furthermore, cows in these experiments known to be susceptible to milk fever were among the most definite demonstrators of the differences in blood and milk calcium specific activity. It is postulated that if such a high specific activity blood calcium fraction does exist, its duration is relatively short after oral dosing. Otherwise, as time progressed the specific activities of blood and milk calcium would diverge markedly r a t h e r than remain parallel, as they have in nearly all of these studies. Additional evidence against a high specific activity fraction of blood calcium is the observation that calcium in the urine and that in blood were n e a r l y identical in specific activity. These results confirm similar observations in dogs (5) and humans (1). If blood calcium fractions of differing specific activities were circulating, it would be unlikely that the kidney would excrete them in the same proportion as that f o u n d in blood. F u r t h e r m o r e , lack of specific activity differences between arterial blood calcium and m a m m a r y vein blood calcium did not indicate that the m a m m a r y gland was removing a high specific activity fraction. Since the total calcium arterial-venous differences in this study were only about 2 to 4% and the peak specific activity differences wl~en exhib.ited were usually of the order of 10 to 15%, the precision of the analytical methods may not have been good enough to show such an effect. Failure to alter milk calcium specific activity as much as that of blood calcium by repeated intravenous doses of calcium suggested that blood calcium recently absorbed largely as ionic calcium may not be preferentially used for milk formation, supposing that absorbed and injected calcium would react similarly. The mass spectrographic analyses of blood and milk calcium from one cow gave no indication of the possibility of a preferential secretion by the mamm a r y gland of heavier than average calcium isotopes. I n an experiment subsequent to the samplings for spectrographic analysis, it was found that this cow (No. 10) did not appear to concentrate Ca 45 in her milk after an oral dose. Until the isotopic analyses can be made on blood and milk of a cow showing the concentrating effect, the possibility of isotope separation in some cows should not be completely rejected. I f there were an isotope separation, however, it would not be expected that the heavy isotopes would enter into the reactions leading to milk formation at a greater rate than the light isotopes. Therefore, this explanation is given v e r y minor consideration.
1606
E.W. SWANSON ET AL
A f t e r the peaks of activity have passed, the specific activity of milk calcium corrected to the average time of secretion of the sample has in a great m a j o r i t y of the experiments remained above the specific activity of blood calcium as long as reliable samples could be collected. The rate of decline of both blood and milk values has been exponential, and the values have been parallel. This situation is consistent with the hypothesis t h a t milk calcium is derived f r o m a homogeneous portion of blood calcium but t h a t a constant delay in the appearance of the milk calcium occurs. This delay can be explained by the existence in the u d d e r of a calcium reservoir f r o m which a large p a r t of the milk calcium is eventually drawn. Such a calcium pool has been suggested by Gowen and Tobey (6) on the basis of excessive ash content of the udder. Monroe (11) found that four udders f r o m cows in late lactation contained two to four times as m u c h calcium as could be accounted for b y the residual milk they contained. I t is possible that udders f r o m cows at the peak of lactation would show even greater stores of excess calcium. Since the total residual milk m a y easily equal 20% of the amount n o r m a l l y secured at a milking, it follows that the recently milked u d d e r m a y contain enough calcium to f o r m the amount of milk to be secreted in the next 12 hours. Another i m p o r t a n t factor in the movement of calcium f r o m blood through the u d d e r to milk is the relatively low exchangeability of milk calcium, which has been demonstrated in these experiments and others (17). I t is postulated t h a t calcium which enters the secretory tissue of the u d d e r is also bound in some m a n n e r so that it does not again r e t u r n easily to the blood but is p r i m a r i l y released or continued in processing to f o r m milk. Such a binding process is necessary to explain the v e r y long periods that have been observed in some cows between equal specific activities of blood and milk calcium. One cow dosed p r e p a r t u m (15) had shown such a lag period of about 10 days for milk secreted d u r i n g the first 10 days of lactation. Several cows in the experiments reported here have shown lag periods of 18 hours or more, and one period was 72 hours. Such long delays cannot be explained by the delay of movement of p r e f o r m e d milk through the udder, as was proposed by L a k s h m a n a n and K u m a r (9). On p u r e l y physical considerations such m~ explanation is not plausible because the total weight of a milked udder is not equal to more than one d a y ' s milk yield. The variability in the time lag between equal calcium specific activities of Mood and milk is p r o b a b l y due to a eombination of m a n y fat'tors. The size of the u d d e r calcium reservoir and its proportionate eontribution to milk calcium is one factor. The data in F i g u r e 8 show that p a r t of the milk ealeimn is derived r a p i d l y f r o m the blood, probabl.v without passing th,'ough the udder reservoir. I f all of the nlilk calcium came d i r e , t l y f r o m blood as tim milk was secreted, the specific activity curves of blood and milk would be expected to coincide in the declining phase, as they have in several of the experiments presented here. I f the calcium binding reservoir of the udder were very large or active, proportionately more of the milk would be derived from this source, which would result in longer lag periods. Another possibility is a variation in exchange-
SECI~ETION OF Ca4~ BY THE MAMMARY GLANDS
1607
ability of udder calcium. The differences observed could be explained without assuming m a j o r changes in the amount of calcium reserve in the u d d e r if exchange rates varied widely. E x t r e m e l y r a p i d exchange would result in a minim u m time lag, whereas a low exchange rate with a given size reservoir would produce a longer period between equality of blood and milk calcium specific activity. More definitive experiments u p o n secreting m a m m a r y tissue are necessary to clarify these questions. SUMMARY
E x p e r i m e n t s have been discussed in which the specific activities of milk calcium and blood calcium have been compared a f t e r intravenous and oral doses of radioactive calcimn administered to lactating d a i r y cows, along with investigations to determine the cause of the differences observed. A f t e r intravenous doses the specific activity curve of blood calcium as it declined passed t h r o u g h the peak of the milk calcium curve corrected to average time of secretion. A f t e r the peak, the milk specific activity usually remained higher t h a n the blood but continued to decline at the same rate as blood. A f t e r oral doses wide variations in response have been observed. I n some animals the specific activity of milk calcium has exceeded the highest observed specific activity of blood, but in others they were nearly equal at their maxinmm. A f t e r the peak the decline curves have been v e r y similar to those a f t e r an intravenous dose. Cows that had previously developed milk fever were f o u n d to exhibit greater differences ill specific activities of blood aud milk calcium in early lactation t h a n other cows; yet these differences were observed also in cows t h a t were not highly susceptible to milk fever. No satisfactory explanation for the excessive p r o p o r t i o n of radioactive calcium in milk has been discovered. P a p e r electrophoresis, arterial-venous differences, and experiments involving the constant injection of stable calcium failed to s u p p o r t the hypothesis t h a t a high specific activity fraction of blood calcium existed which m i g h t be used p r e f e r e n t i a l l y for milk formation. Mass spectrographic analysis of blood a n d milk calcium of one cow showed no differences in proportions of six calcimn isotopes, which indicated that the u d d e r was not concentrating Ca 45 per se. The existence in the u d d e r of a variable calcium reservoir f r o m which the calcium is not readily exchanged with blood calcium, and which is used for milk formation, has been postulated to explain the differences in time between equal specific activity values of blood and milk calcium. ACKNOWLEDGMENTS T h e t e c h n i c a l a s s i s t a n c e of I r a W h i t n e y , G. W. R o y s t e r , a n d T. G. Clark in m o s t of t h e s e e x p e r i m e n t s is g r a t e f u l l y acknowledged. T h e n m s s s p e c t r o g r a p h i c a n a l y s e s of blood a n d milk c a l c i u m were done b y Oak R i d g e N a t i o n a l L a b o r a t o r y t h r o u g h t h e c o u r t e s y of R. F . t t i b b s . REFERENCES ( ] ) BLAU, M., tion of (2) COMAR, C. U s e of
SPENCER, H., S~VERNOV, J-., AND LASZLO, ]). U t i l i z a t i o n a n d I n t e s t i n a l ExcreC a l c i u m in M a n . Science, 120: 1029. 1954. L., HANSARD, S. L., HOOD, S. L., I:)LUMLEE, M. P., AND BARRENTINE, B. F . Calcium-45 in Biological Studies. N~lcleonics, 8: 19. 1951.
1608
E. W. S W A N S O N E T A L
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