HISTOLOGICAL studies of the changes which occur in the mammary gland of. began when the pressure in the mammary gland reached 25 mm. Hg.

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1 THE ABSORPTION OF SERUM ALBUMIN AND CASEIN FROM THE MAMMARY GLAND OF THE MERINO EWE. By A. K. LASCELLES. From the Department of Experimental Pathology, John Curtin School of Medical Research, Australian National University, Canberra. (Received for publication 14th July 1961) Experiments have been carried out to determine the way in which 131I human serum albumin (131I HSA) and 131I casein are removed from the mammary gland when milk is allowed to accumulate in the gland. Lactating merino ewes with the mammary lymphatic duct cannulated were used in these experiments. The labelled proteins were introduced into the mammary gland through the teat duct and milk was allowed to accumulate in the gland. Lymph, plasma and urine samples were collected during the next 7-14 days. The results suggested that 131I HSA was absorbed from the mammary gland without being broken down and was removed largely by way of the lymphatics. It also appeared that casein was absorbed from the mammary gland and entered the interstitial pool without being broken down. However, the casein was rapidly transferred from the interstitial fluid into the blood stream. 131I casein when introduced intravenously into lactating ewes rapidly left the blood and entered the lymph, milk and urine suggesting that the casein in the blood existed as a small molecule. The significance of these results is discussed in relation to the physico-chemical forms in which casein occurs in the body fluids. HISTOLOGICAL studies of the changes which occur in the mammary gland of rats when milk is allowed to accumulate have been made by a number of workers [Silver, 1956; Jeffers, 1935]. Obvious signs of involution were not seen until at least 3 days after suckling had ceased. Peterson and Rigor [1932 a] demonstrated that milk secretion in the cow ceased and absorption began when the pressure in the mammary gland reached 25 mm. Hg. It was also shown that absorption greatly altered the composition of milk. A fall in the concentrations of lactose, fat, calcium, phosphate and an increase in the concentration of total protein and ash in the milk was reported by Peterson and Rigor [1932 b]. The udder of a well-fed merino ewe in early lactation will produce approximately 1.5 litres of milk a day [Davies, 1958]. If suckling is stopped at the height of lactation, large amounts of milk fat, casein, whey proteins and lactose, substances which are not normally found elsewhere in the body, will accumulate in the mammary gland. During the next 7-14 days most of the milk which has been secreted will be removed from the udder. The volume of milk remaining in the udder after normal weaning is less and removal of this milk may occur more slowly. Lascelles [1961] reported that the lymph draining the mammary gland became quite opalescent in character when the gland became acutely distended with milk. The opalescent lymph was found to contain high concentrations of calcium, inorganic phosphate and esterified fatty acids. Milk 48

2 The Absorption of Proteins from the Mammary Gland proteins were identified in the opalescent lymph by immunological techniques. No significant change in the composition of the lymph was observed unless the udder became acutely distended. Phosphate and acetate have been shown to be absorbed from the milk after injection into the udder cavity [Kleiber and Luick, 1956; Rogers and Kleiber, 1957]. Azimov [1958, 1959] found that radioactive calcium and phosphate were readily absorbed from the milk in goats and cows. The rate of absorption increased as a result of milking or massage. An increased rate of absorption of radioactive phosphate was also found to occur when milk was allowed to accumulate in the gland. Following the introduction of casein, labelled with radioactive phosphate into the udder cavity, radioactivity was detected in the bloodstream in inorganic form. This paper describes the results of experiments which were carried out to establish the way in which human serum albumin (HSA) and casein are removed from the mammary gland of merino ewes. MATERIALS AND METHODS Merino ewes in early- to mid-lactation were used in the experiments. Mammary lymphatic ducts were cannulated with plastic ("Transflex") tubing as described by Lascefles and Morris [1961]. Labelled Serum Albumin.-Radioactive iodinated human serum albumin (1311 HSA) was obtained from the Radiochemical Centre, Amersham. Fractionation and Labelling of Casein.-The ewe's milk was centrifuged at 3000 r.p.m. for 30 min. at 0-5 C. and the cream removed. The skim-milk was The casein precipitate brought to a ph of 4-5 by slowly adding 1 N hydrochloric acid. was separated from the whey by centrifugation, washed three times with distilled water and then redissolved by suspending the washed precipitate in a small volume of distilled water and adding 0-5 N sodium hydroxide until a ph of 7 0-7*3 was obtained. The procedure of acid precipitation and washing was repeated and the casein again was dissolved by the addition of alkali. 5 ml. of the casein solution containing approximately g. casein were made up to 10 ml. by the addition of 4 ml. calcium magnesium saline (sodium chloride 8-5 g., anhydrous calcium chloride g., magnesium chloride g., sodium borate g., boric acid g., distilled water to 1 litre) and 1 ml. of freshly prepared 0-2 M sodium carbonate-bicarbonate buffer (ph 10). Approximately 0-25 mc. carrier-free 131J was added to a test tube containing 2-0 ml. carbon tetrachloride, 2-5 ml. of 10-4M potassium iodide and 0 05 ml. 2 N sulphuric acid. 0-2 ml. hydrogen peroxide was added and the two phases were shaken for 30 min. Both carrier and radioactive iodine were liberated and were taken up in the carbon tetrachloride. The water phase was then removed by aspiration, leaving the slightly pink carbon tetrachloride which contained the carrier and radioactive iodine. The carbon tetrachloride phase was washed twice with 10 ml. of distilled water. The casein solution was then added to the carbon tetrachloride-iodine solution and the two phases were mixed very gently for 10 min. The casein solution was centrifuged to remove any insoluble matter. The solution was dialyzed, first against running water for 12 hr. (to remove the free iodine) and then against calcium magnesium saline for hr. [Webster and Laver, 1961] labelled casein of a satisfactory specific activity was obtained by the use of this method. Electrophoresis on filter paper in barbiturate buffer, ph 8-4 showed two fractions (a- and /3-casein) which were radioactive. This procedure did not produce uniform results as some preparations contained a varying percentage of apparently denatured protein. The VOL. XLVII, NO

50 Lascelles suitability of each preparation was tested by injecting a tracer dose intravenously into a sheep to establish the behaviour of the labelled material in the circulation.

3 50 Lascelles suitability of each preparation was tested by injecting a tracer dose intravenously into a sheep to establish the behaviour of the labelled material in the circulation. Of a total of twelve preparations, two were found to contain a high proportion of denatured protein. Following the intravenous injection of these apparently denatured preparatiolns it was found that most of the radioactivity was removed from the circulation in the first 2-3 min. and after 10 min. the radioactivity remaining in the plasma was not bound to protein. The doses of 1311 HSA and of 1311-labelled casein were adjusted so that approximately 6 x 106 counts/min. were introduced into the mammary gland. The radioactive protein was injected into the empty mammary gland cistern through a sterile polythene tube. Milk was then allowed to accumulate in the gland and plasma and lvmph and urine samples were collected during the next 1-2 weeks. A dose of radioactive casein containing approximately 2 x 106 counts/min. was injected intravenously into each of three lactating ewes with lymphatic duct fistulae. The radioactivity in the plasma, lymph, milk and urine was measured during the next 24 hr. Radioactive measurements were made at the completion of each experiment. The samples (0-2 ml.) were plated onto plastic planchets and evaporated to dryness. The protein in 02 ml. samples of plasma, lymph and milk was precipitated by adding 5 ml. of 7-5 per cent trichloracetic acid and the precipitate was collected on discs of filter paper. The supernatant fluid obtained after centrifugation of the trichloracetic acid precipitate of a 0-2 ml. sample was spread and dried on a planchet. Trichloracetic acid precipitates and filtrates of urine samples were obtained after the addition of carrier bovine albumin to the urine. The casein was isolated from one of the milk samples using the method described by Barry [1952]. Approximately 1 per cent solution of the purified casein was made and the radioactivity of a 0-2 ml. sample was measured. All radioactive measurements were made with a thin mica end-window G.M. tube. RESULTS Removal of Albumin.-After the introduction of 131JI HSA into the mammary gland cistern, radioactivity appeared in the lymph; the level of radioactivity increased to a maximum and then fell exponentially. The time course of the appearance of the radioactivity varied considerably and seemed to be related to the degree of distension of the mammary gland. Figs. 1 and 2 show the rate of removal of the radioactivity from the mammary gland by the mammary lymphatics in a ewe in early lactation and in a ewe in mid-lactation respectively. It can be seen that the mammary lymph of sheep in early lactation contained high levels of radioactivity hr. after the introduction of the radioactive material. Since all the radioactivity was bound to protein it appeared that the 1311-labelled HSA was removed from the mammary gland without being broken down. The percentage of the original activity collected in the lymph and urine and the estimated percentage of the original activity present in the total body extracellular fluid at the end of the experiment are shown in Table I. The radioactivity in the plasma, which was always very low relative to that in lymph, was bound to protein. The radioactivity which appeared in the urine however was not precipitated with trichloracetic acid and was presumably in the form of iodide or iodo-tyrosine.

4 The Absorption of Proteins from the Mammary Gland on 0 0 u DASATE NROUTO 1 A 5 UU z Qd 0i 2 z DASATRITOUTO 14 DAYS AFTER INTRODUCTION FIG. 2.-The percentage of the original radioactivity collected in the lymph following the introduction of 13lI HSA into the mammary gland cistern of a ewe in mid-lactation.

5 52 Lascelles Removal of Casein.-After 131I casein was introduced into the gland, a low level of radioactivity often appeared in the mammary lymph after hr. The level of radioactivity in the plasma increased and after hr. was equal to, or even slightly exceeded, the level in the lymph. Table II shows the results of one of five experiments. The radioactivity in TABLE I.-THE PERCENTAGES OF THE ORIGINAL RADIOACTIVITY, COLLECTED IN THE LYMPH AND URINE AND THE PERCENTAGE REMAINING IN THE TOTAL BODY EXTRA- CELLULAR FLUID FOLLOWING THE INTRODUCTION OF 131I HSA INTO THE MAMMARY GLAND CISTERN OF LACTATING EWES. Percentage original activity Experiment In extracellular Total Collmphted Colle fluid at end of recovered experiment both the plasma and the lymph was almost entirely bound to protein. However, in one experiment which was conducted over a period of 14 days the appearance of significant radioactivity which was not bound to protein was observed on the 13th and 14th days. In one experiment in which the udder became grossly distended with milk, the lymph suddenly became opalescent 32 hr. after suckling was stopped. The radioactivity in the TABLE II.-THE RADIOACTIVITY IN PLASMA AND LYMPH FOLLOWING THE INTRODUCTION OF CASEIN 1311 INTO THE MAMMARY GLAND CISTERN OF A EWE IN MID-LACTATION Radioactivity Radioactivity Hr. after (counts/min.) Hr after (counts/min.) injection injectiohn 0-2 ml. sample r..min 0-2 inmjection ml. sample A- Lymph Plasma Lymph Plasma opalescent lymph was times that of the lymph collected in the 31st hr. and as the opalescence subsequently decreased the radioactivity decreased. The percentage of the original radioactivity introduced into the mammary gland cistern which was collected in the lymph and urine and which was present in the extracellular fluid at the end of the experiment, is given in Table III. The results show that the lymphatics played a minor role in removing the radioactive casein from the mammary gland. Approximately half the radioactivity in the urine was found to be bound to protein throughout the first 7 days of the experiment. The proportion of radioactivity bound to protein decreased during the next few days.

6 The Absorption of Proteins from the Mammary Gland The level of radioactivity in the plasma and in the mammary lymph of a ewe following the intravenous injection of 131I-labelled casein is shown in fig. 3. The radioactivity disappeared from the plasma rapidly; the half-life TABLE III.-THE PERCENTAGES OF THE ORIGINAL RADIOACTIVITY, COLLECTED IN THE LYMPH AND URINE AND THE PERCENTAGE REMAINING IN THE TOTAL BODY EXTRA- CELLULAR FLUID FOLLOWING THE INTRODUCTION OF CASEIN 131I INTO THE MAMMARY GLAND CISTERN OF LACTATING EWES. Percentage original activity Experiment Collected Collected In extracellular Total in lymph in urime fluid at end of experiment recovered being a little less than 30 min. The radioactivity appeared rapidly in the lymph and after approximately 1 hr., the level in the plasma and the lymph was equal. Between 70 and 90 per cent of the radioactivity in the plasma and lymph was bound to protein. Very high levels of radioactivity were >140 -\ o / S HOURS AFTER INJECTION FIG. 3.-The radioactivity in the plasma and lymph (counts per min.) of a ewe in mid-lactation following the intravenous injection of casein 131I. found in the milk collected after the 1311-labelled casein was injected intravenously and it was estimated that per cent of the injected radioactivity was removed by the mammary gland and secreted into the milk. Significant levels of radioactivity were found in the milk samples collected 30 min. after the intravenous injection. The maximum levels of radioactivity in the milk 53

54 Lascelles occurred after 2-3 hr. and these were 3-5 times greater than the highest level in the plasma. All the radioactivity during the first 2-3 hr. was bound to protein.

7 54 Lascelles occurred after 2-3 hr. and these were 3-5 times greater than the highest level in the plasma. All the radioactivity during the first 2-3 hr. was bound to protein. The casein in a milk sample which was collected 2 hr. after the intravenous injection in one of the sheep was fractionated and it was concluded that all of the radioactivity in the milk sample was associated with casein. A decreasing proportion of radioactivity bound to protein was found in the milk collected between 3 and 24 hr. after the injection. DISCUSSION The results indicate that the epithelium within the mammary gland is permeable to human serum albumin. Once the albumin found its way into the interstitial fluid it was taken up by the regional lymphatics. It is known that one of the functions of the lymphatic system is to return protein which collects in the interstitial fluid to the general circulation [Yoffey and Courtice, 1956]. Thus the mammary lymphatics seem to play an important role in the return of albumin which has been transferred from the milk to the interstitial fluid, to the general circulation. The low level of radioactivity which was bound to protein was found in the plasma of two sheep towards the end of the experimental period. The rate of flow of lymph diminished rather rapidly in these sheep and this was thought to be due to clots forming within the plastic tube. This in turn may have caused a back pressure of lymph which would lead to the development of collateral lymphatics [Carlston and Olin, 1952]. The difference in the rate of removal of albumin by the mammary lymphatics in the two experiments shown (figs. 1 and 2) was thought to be due at least in part to the difference in the degree of distension of the mammary glands in the two sheep. In the first example (fig. 1), in which a large proportion of the albumin was removed in the first 72 hr., the udder became very distended with milk and the lymph became opalescent in character 24 hr. after the experiment began. The pressure within the ducts and the acini would be maximal under these conditions and this may have caused an increased rate of transfer of albumin into the interstitial pool. Some acini or ducts may actually burst under these conditions [Selye, 1934; Lascelles, 1961] and this may be important in causing the early appearance of radioactivity in the lymph. It was not possible to account for more than 70 per cent of the radioactivity introduced. This was due to the fact that a significant amount of radioactivity still remained in the gland at the end of the experiment. Some of the I 131HSA which entered the blood would be metabolized and a proportion of the free iodide released would be taken up by the thyroid gland and would not be measured. After the introduction of l31i casein into the mammary gland radioactivity appeared in the lymph, and at hr. the level of radioactivity was equal in both plasma and lymph. The radioactivity in the plasma and lymph was bound to protein and it seems that the 1311-labelled protein was casein. Moreover, the results show that the '31I-labelled casein was not absorbed to

The Absorption of Proteins from the Mammary Gland any extent by way of the lymphatic system and this suggests that it was removed from the mammary gland in a form which was able to enter the blood

8 The Absorption of Proteins from the Mammary Gland any extent by way of the lymphatic system and this suggests that it was removed from the mammary gland in a form which was able to enter the blood stream directly. These results are at variance with those of Azimov who found only inorganic label in the blood following the introduction of labelled casein into the cavity of the mammary gland. Azimov does not appear to have repeated this experiment nor did he carry out any experiments to determine the behaviour of the 32P casein in the circulation after its intravenous injection. The casein in milk has been shown to occur in various physico-chemical forms. Most of the casein is present in the form of micelles which vary in size between 30 myu and 300 my [Nitschmann, 1949; Hostettler and Imhoff, 1951 a and b]. A small proportion of the casein is also thought to exist in the forms of small aggregates and monomers. The three physico-chemical forms are presumed to be in equilibrium. The importance of calcium in the formation of the micelles is well recognized and the alteration of the distribution of casein between the soluble and micellar forms by the removal and addition of calcium has been described by von Hippel and Waugh [1955]. The resolution of acid casein by moving boundary electrophoresis into a-, 3- and y-peaks was observed by Mellander [1939]. More recently a new component calledk-casein has been identified [von Hippel and Waugh, 1955; Waugh and von Hippel, 1956]. These workers described K-casein as occurring in skim milk in the a-k-complex. It is generally agreed that at room temperature and neutral ph in the presence of salt a- and /3-casein are aggregated. As the ph is raised the casein disaggregates and at ph the casein is found in monomeric form [McMeekin and Peterson, 1955; von Hippel and Waugh, 1955]. Likewise K-casein is highly aggregated at neutral ph and is disaggregated into monomers at ph 12 [Wake, 1959]. Molecular weights of a- and /-casein are approximately 25,000 and 17,000 respectively [MacKenzie and Wake, 1959 (III)]. The molecular weight of the K-casein monomer was found to be approximately 26,000 [Wake, 1959]. It would appear that the 1311-labelled acid casein preparations used in the experiments described in this paper were in the physico-chemical forms of monomers and aggregates. Their physico-chemical forms following introduction into the mammary gland would change. At the lower ph of milk and in its characteristic electrolyte environmlent the casein would be expected at least to aggregate more heavily and it seems probable that much of it would form micelles. Thus the '311-casein which was probably in the form of large aggregates and micelles after introduction into the mammary gland, appeared to be absorbed as small protein molecules. These may have been casein monomers. It is significant in this regard that after 131I casein was injected intravenously, it left the plasma rapidly and the radioactivity in plasma and lymph was equal 1 hr. after the injection. These results demonstrated the rapid transfer of the 131I casein preparation and suggest that the casein in the circulation was present in the monomeric form. The fact that high levels of 131I casein occurred in the milk following its intravenous injection also showed that 55

9 56 Lascelles the casein in the circulation readily entered the mammary gland. It also suggests that after the casein molecule had diffused into the milk, with its low ph and its characteristic electrolyte environment, it was sequestered there as aggregates or micelles. It may be that as the calcium concentration of the milk decreases and as the ph increases during the period in which milk is absorbed from the gland [Peterson and Rigor, 1932 b] there is a gradual alteration in the physico-chemical forms of casein in the milk. The equilibrium reaction, casein micelles - casein aggregates < casein monomers, would tend to favour the formation of casein monomers. The casein monomer diffuses into the interstitial fluid of the mammary gland and from there into the general circulation. The rate of absorption of both casein and albumin from the mammary gland is slow and appears to be related to the bulk movement of fluid. ACKNOWLEDGMENTS The encouragement and advice of Dr. Bede Morris was greatly appreciated. Invaluable technical assistance was given by Mrs. J. de Waal and Mrs. H. Kobau. REFERENCES AzIMov, G. I. (1958). Peaceful Uses of Atomic Energy, Section 27, 138. Proc. 2nd U. N. Inter. Conf. AzIMov, G. I. (1959). Priroda, p. 50. BARRY, J. M. (1952). J. biol. Chem. 195, 795. CARLSTON, A. and OLIN, T. (1952). Acta. Physiol. Scand. 25, 260. DAVIES, H. L. (1958). Proc. Aust. Soc. Anim. Prod. 2, 15. JEFFERS, K. R. (1935). Amer. J. Anat. 56, 257. VON HIPPEL, P. H. and WAUGH, D. F. (1955). J. Amer. Chem. Soc. 77, HOSTETTLER, H. and IMHOFF, K. (1951 a). Milchwissenschaft, 6, 351. HOSTETTLER, H. and IMHOFF, K. (1951 b). Milchwissenschaft, 6, 400. KLEIBER, M. and LUIcK, J. R. (1956). Ann. N.Y. Acad. Sci. 64, 299. LASCELLES, A. K. (1961). Nature, 191, LASCELLES, A. K. and MORRIS, B. (1961). Quart. J. exp. Physiol. 46, 199. MCKENZIE, H. A. and WAKE, R. G. (1959). Aust. J. Chem. 12, 734. MCMEEKIN, T. L. and PETERSON, R. F. (1955). Abstr. 128th Meeting American Chemical Society, Minneapolis, 2A. MELLANDER, 0. (1939). Biochem. Z. 300, 240. NITSCHMANN, H. (1949). Helv. Chim. Acta. 32, PETERSEN, W. E. and RIGOR, T. V. (1932 a). Proc. Soc. exp. Biol. Med. 30, 254. PETERSEN, W. E. and RIGOR, T. V. (1932 b). Proc. Soc. exp. Biol. Med. 30, 257. ROGERS, T. A. and KLEIBER, M. (1957). Proc. Soc. exp. Biol. Med. 94, 705. SELYE, H. (1934). Amer. J. Physiol. 107, 535. SILVER, I. A. (1956). J. Physiol. 133, 65P. WAKE, R. G. (1959). Aust. J. biol. Sci. 12, 538. WAUGH, D. F. and VON HIPPEL, P. H. (1956). J. Amer. Chem. Soc. 78, WEBSTER, R. G. and LAVER, G. W. (1961). Aust. J. exp. Biol. and Med. Sci. [To be published.] YOFFEY, J. M. and COURTICE, F. C. (1956). Lymphatics, Lymph and Lymphoid Tissue. London: Edward Arnold & Co. Ltd.

y,-globulin which is present in high concentration in colostrum. Subsequently, flow

y,-globulin which is present in high concentration in colostrum. Subsequently, flow Q. Jl exp. Physiol. (1968) 53, 415-421 LYMPH FLOW AND PROTEIN COMPOSITION OF THORACIC DUCT LYMPH IN THE NEWBORN CALF. By A. D. SHANNON and A. K. LASCELLES. From Dairy Research Foundation, University of