STUDIES ON (a) me paecunsons or MILK worms IN THE RABBlT AND (2:) THE HORMONAL REQUIREMENTS or GUINEA ms MAMMARY TISSUE m w Thesis 96!! the Degree at pk. o. MICHEGAN STATE UNWERSITY George Cayman? Gerrifsen E969 This is to certify that the thesis entitled STUDIES ON(a) THE PRECURSCRS OF MILK PROTEINS IN THE RABBIT AND (b) THE HORMUNIL REQUIREMENTS OF GUINEA PIG NAMMARY TISSUE £3 VITRo presented by George Contant Gerritsen has been accepted towards fulfillment 9f the requirements for __Ph.D.._ degree in .Physinlogy IJBRARY' Michigan State University STUDIES 0:: (a) T113 R1130: .3011. T 01“ 1.1111: R: 0'11 1:113 :11 1:1: 11:.1—11721'1' AND (b) m; ROI-1:01:11, 11:31:: 5:33:73 OF GUI: .- 1:. PIG ‘,""Itfi' ”or” Q. ynmtr' adu‘nuuus Iiup-JIL S’s-..o By George Content Gerritsen Ml LLCTI -CT Suhzfitted to the School of Advanced Graduate Studies of Ifichigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PEIIIDSOPIEY Department of stiolow and Pharmacology 1960 7 "7 Approvd ’W k (“I Ljifj / 3 L LVC C it?!) x/ «7 ABSTRACT GEORGE C. GERRITSEN l thssiixsa 1. The objectives of this research were: a. To provide quantitative data on the precursors of caseins, B-IactogIobuIin and O<~lactalbumin of rabbit milk. b. To provide quantitative data on the relationship between Y'-g1obu1ine of blood and immune globulins of rabbit milk. c. To provide quantitative data on the relationship between albumin of blood and the "blood" almmin of rabbit milk. d. To deve10p a synthetic medium enriched with hormones which would be capable of maintaining non-secretory mamasry tissue in m. e. To develop e medium enriched with hormones capable of initiation of lactation in non-secretory mannnary tissue in m. f. To develop e medium enriched with hormones capable of main- taining secretion in secretory mammary tissue in m. 3. To study the ability of secretory mammary tissue to take up amino acids and blood proteins in mm. MM: 1. The methods utilized in this research involved the blood fractionation by the procedures or Cohn gt 2.1- (1950). Albumins, Y-globulins and crude fractions of B-globulins and t><--glomlins were obtained by this method. Paper electrophoresis patterns were run on these fractions and on whole serum to prove their identity and establish their homogeneity. 2. Two male New Zeeland white rabbits were injected with cl“ GEORGE C. GERRITSEN 2 labeled B51003. The animals were placed in a chamber designed and built to trap expired cl“ labeled C02. Six hours post injection, the animals were bled and Cl“ labeled serum protein fractions were isolated by the method or Cohn rt 31. (1950). 3. Classical ammonium sulfate fractionation procedures were em— ployed for the fractionation of the fi -lactoglobulin. od-lactalbumin and "blood" albumin in rabbit milk. Rabbit milk caseins were precipi- tated by adjusting the pH to their isoelectric point which was found to be “.3. It was found that the imune globulin fraction of rabbit milk could be isolated by readjustment of the acid whey to pH 6.0. A method for paper electmphoresis of rabbit whey and rabbit milk pro- teins was deveIOped which gave very good results. This method was the precoating of the paper strips with gelatin, thus preventing the absorption of the whey proteins into the paper strips. 1+. The percent of leucine and glutamio acid were determined in the blood protein and milk protein fractions. This was accomplished by hydrolysis of the proteins and by column chromatography. The eluents from the column were subjected to paper chromatography as a purity check. Aliquots of eluents were treated with ninhydrin and the amount of amino acid determined colorimetricaIJy. Free blood leucine and glutamic acid were isolated from the blood by column chromatography. The chemical purity of Dlpleucine-Z-Cll’ and DL-glutamic scid-Z-Clh was established by paper chromatography and subsequent counting of the paper strips in a gas flow strip counter. 5. A method was deve10ped for the milking of rabbits. This was GEORGE c. GERRITSEN 3 accomplished by injecting 1.0 LU. of ontocin into the marginal ear vein. A beveled glass tube was placed over the nipple imediately following the injection. The tube was connected to a reservoir for collection of milk which was in turn connected to a water aspirator. A negative pressure of 15 m. of Hg. was maintained in the system. Vigorous massage of the marmary gland from the periphery toward the base of the nipple was applied together with periodic interruption of the suction by partial removal of the tube from the nipple. Blood samples were obtained from the rabbits by heart puncture into the left ventricle. 6. Dlpleucine-Z-Clu', DL-glutamic acid-2.01“ and citprotein fractions were dissolved in a minimal amount of phosphate buffer (pH 7.2) and injected by marginal ear vein into 10 lactating rabbits. Blood and milk samples were obtained at 2, 6, 12, 21+, 36, 1+8 and 72 hours post injection. The blood and milk samples were fractionated into the various serum and milk protein fractions described above. 7. The blood and milk protein fractions were dialysed against dis- tilled water until free of reagents. The fractions were then lyophil- 1180a. Glutamic .oid was isolated from the protein fractions by acid hydrolysis and precipitation of the chloride of glutamic acid. The protein. free glutsmic acid and leucine samples were counted by a ‘liquid scintillation counting procedures. W 1. In kperiment l. 45 uc. of DLleucine-Z-Cll" was injected into ‘ luctating rabbit on the lath day postpartum. The specific activity IL .‘ GEORGE C . GERRITSEN u. of leucine was determined in: serum, Y-globulins, [3 ~globulins, 0(-globulins, albumin, caseins, inmune globulins. c( -l.actalbumin, a-lactoglobulin and "blood" serum albumin. The specific activity of free blood leucine decreased at an extremely rapid rate during the first 10 minutes post injection. The calculated value at zero time was 157.2 uc./mm. in the extracellular water. At 10 minutes, the ex- perimentally determined value was 3.7 uc./mm. The equation for this decrease in the specific activity of free blood leucine is A=Aoe'o°375t. The ti value is 1.85 min. and the rate constant is 0.375 min.’1. After 10 minutes the rate constant changes very drastically to a value of -o.00169 mil with a ti. of uo9.5 min. A system to explain this finding has been postulated. 0n the basis of these data and the shape of the curve. it has been postulated that the leucine injected has been sequestered somewhere in the animal's body. Likely possibilities are the reticulo-endothelial system, the intracellular pool and plasma pro- tein binding. After two hours the leucine from this sequestered pool appears to return to the extracellular leucine pool at a rate slightly less than the rate of removal of free leucine from the extracellular pool. Thus . it appears that only a small proportion of the injected Brendan—2-014 was available for synthesis into milk proteins at any particular time. Thus. an attempt was made to establish the parameters for leucine as a chireet precursor of milk proteins. It appears that the curve for free blood leucine as determined in Experiment 1 represents the amount of free blood leucine available for incorporation into milk proteins. GEORGE c. Gasman}: 5 2. The data in EXperiment 1 show that the leucine incorporated into caseins, fl -lactoglobulin and oz-lactalbumin of rabbit milk are derived from the free blood leucine. The curves fit the criteria for precursor product relationships very well. Further, it takes an average of it hours for a free blood leucine molecule to be incorporated into a milk protein and transferred to the ducts ready for excretion as a milk constituent. 3. The ratios of the specific activities of blood I" -globulins and milk imune globulins in Elcperiment 1 show that at least 73.5% of the milk immune globulins were derived directly from the blood Y-globulins. The immine globulins of the milk were not synthesized I within the mammary gland since the specific activities of leucine incorporated into the immune globulins of milk and the specific activi- ties of caseins. B -lactoglobulins and M-lactalbmnins are different. A. It is also concluded from Ebcperiment 1 that at least 77.1% of the "blood" serum albumin in rabbit milk is derived directly from the albumin of blood plasma. This is based on the ratios of the specific activity in these 2 protein fractions. The blood Y-globulins and albumin rapidly achieve equilibrium with the milk immune globulins and ”blood“ serum albumin of milk. The ratio of the specific activity of the immune globulin from milk to Y—globulin from blood at 2 hours post injection was 0.916 and the ratio of specific activity of "blood" 801mm albumin from milk to albumin from blosd at 2 hours post injection was 0.900. thus demonstrating the rapid equilibrium between these blood Pmteins and their corresponding milk proteins. GEORGE C. GERRITSEN 6 5. Experiments 2. 3 and u with DL-leucine-Z-Clu and DL—glutandc acid-Z-Cll‘ completely substantiate the results on the first experiment with mpleucine.2.c1‘*. It is noteworthy that all of the glutamic acid incorporated into caseins. B-lactoglobulin and o<~lactalbumin of the milk of lactating rabbits apparently comes directly from the free blood glutamic acid. 6. kperiments 5 and 6 were designed to evaluate the importance 'of plasma proteins as precursors of milk proteins by the injection of Cl“ hbeled . -plasma protein fractions into lactating rabbits. When cl“ labeled Y-globulins were injected into 2 Lactating rabbits. a close correlation was obtained between the specific activities of the Y-globuline and milk immune globulins. The time required to establish equilibrium between the blood and the milk fractions was slightly more than 2 hours in one animal and slightly more than 6 hours in the other animal. After equilibrium had been reached. it was calculated from the ratios of the specific activities of milk irmnine globuline to Y-globuine that 93.1% of the milk imune globulin in the milk of one animal and 98.2% of milk imme globulin in the milk of the other animal were derived from the Y-globulin in the plasmas. 7. when c1“ labeled albumins were injected into’z lactating rabbit. in Experiments 7 and 8, the relationship between the plasma albmins and ”blood“ serum albumin in milk was found to be similar to that for Y-globulins and milk immune globulins. In this case, it took between 6 and 12 hours to establish an equilibrium between the blood plasma albumin and the "blood" serum albumin from milk. This was true in both animals. GEORGE C. GERRITSEN 7 After equilibrium had been established. it was calculated from the ratios of the specific activities of "blood” serum albumin in milk to the specific activities of albumin in plasma that 92.2% of the "blood“ serum albumin in the milk of one animal and 98.2% of the "blood" serum albumin in the milk of the other animal were derived from the albumin in the plasma. 8. Experiments 9 and 10 on the injection of cl“ labeled o(- and B-globulins from blood plasma indicated that they were of little importance as precursors of milk proteins. Also. no milk protein fraction was detected which was derived directly from these two blood protein fractions. Wtudies with Manmagy Tissue Cultures l. Mamary tissue was cultured by the organ culture method. The axplanta were placed on treated rafts of cellulose-acetate. The rafts were floated in the synthetic medium in a watch glass in a Petri dish. The Implants were cultured for 5 days. The medium was changed when the pH dropped below 7.0. Histological sections of the explants were prepared ani stained with iron hemotowlin and eosin in the usual manner. 2. Media for mamary tissue culture were prepared from Parker's ”199' synthetic medium. Prolactin, l'ydrocortisone and insulin were added at 2 concentration levels . The levels of these hormones for mammary tissue maintenance were 140, 8 and 70 ug./ml. of medium, respectively. The levels of these hormones for maintenance of secretion in may tissue were 21K). 16 and 140 pg./ml. of medium. reapectively. The media were gassed with 95% 02 and 5% cog. DL-leucine-Z-Cll‘, GEORGE C . GERRITSEN 8 DL-glutamic acid—2-014. cl“. Y—globulin and alkalbtmdm were also added to portions of the media for maintenance of secretion in m. 3. Autoradiographs were prepared from eXplsnts cultured in media containing Cl“ labeled amino acids or proteins. The sections of the tissues were mounted on microscope slides and stained with hematoxylin and eosin in the usual way. The slides were coverslipped with celloidin. A thin film of Eastman Kodak emulsion was applied in the darkroom. The slides were sealed in a light proof container and placed in the refrigerator for exposure. The exposed autoradiographs were developed with dektal, fixed in acid fix and washed with tap water. They were then dehydrated and coverslipped in the usual manner. 1+. It was shown that Y-globulin can be taken up by mammary tissue in active secretion and concentrated in the alveolar cells in £11.29. This ability is destroyed by heating the explant to 50°C Just prior to cultivation. 5. Prolactin (2‘40 pg./ml.), hydrocortisone (l6 ug./ml.) and insulin (luo pg./m1.) added to a synthetic medium are capable of main- taining active secretion in namely tissue in nm. This was shown by histological preparations and by the active uptake of leucine and glutamic acid and their apparent incorporation into mammary secretory products. 6. Albumin is not taken up by the secretory mamnary tissue in 111429.. 7. Heating explants to 50°C for 1 minute destroys the ability of unwary tissues to take up Y -globulins. leucine and glutamic acid 1.3mm. GEORGE C . GERRITSEN 9 8. Prolactin (lho‘ug./ml.). hydrocortisone (8 ug./ml.) and in- sulin (70 pg./ml.) in a synthetic medium.are capable of maintaining nonnaeoretory'mammary tissue inyzitng. Seventyathree percent of the total lobulo-alveolar tissue was maintained in 108 emplants prepared from 8 non-lactating guinea pigs. 9. Prolactin (sue ug./ml.). hydrocortisone (16 pg./ml.) and insulin (70 ug./ml.) in a synthetic medium are capable of maintaining secretory mammary tissue in.yi:nn. Seventy percent of the total lobulo—alveolar tissue was maintained, and 55.h¢ of the total lobulo- alveolar system‘was maintained in an active secretory state in 180 explants prepared from 12 lactating guinea pigs. 10. The data obtained on milk immune globulins and "blood" serum albumin in milk and the data obtained by the addition of cl“ labeled dYLglobulins and albumins to tissue culture media indicate that there is a different mechanism.by which the mammary epithelium incorporates these proteins into milk. It is postulated that the mammary gland actively takes up -globulins or is permeable to -globulins, and that it does not actively take up plasma albumin or that the mammary gland is relatively impermeable to albumin. STUDIES ON (a) THE PRECURSORS OF MILK PROTEINS IN THE RABBIT AND (b) THE HORMONAL REQUIREMENTS OF GUINEA PIG MAMMARY TISSUE IN VITRO By George Contant Gerritsen A THESIS Submitted to the School of Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology and Pharmacology 1960 ..-.-0. ~‘ . on- ‘ i L/ / I"! // TABLE OF CONTENTS Page INTRODUCTION -_nmnm“1. .111“ .11111._.“_111_11111_ 1 33qu OF LITERATURE _-.._..__...-. W- 1.1.1.1.“-.. L; I. The Biosynthesis of Milk Proteins —m-~w——w-m~w-~mmw-w-_- u A. Techniques Utilized for the Study of Milk Protein Precursors and the Results Obtained 111.1-1-111-111-_ 4 1. Arterio-Venous Differences in the Concentrations of Amino Acids and Proteins of Blood -1 ---------- 4 2. Perfusion and Tissue Slice Studies on Mammary Glands -—-mmu-m--~~-« --~~-~—-ww~~-w-~----~- 9 Be The Use of Isotopes for the Investigation of the Precursors of Milk Proteins ~ 10 h. The Source of Immune Globulins and "Blood" Serum Albumins in Milk -~- 12 5. In git 1.2 Cultivation of Mammary Tissue --------- 14 MATERIAIS AND METHODS - l6 A. Methods Utilized in the 311111.35 of Rabbit Milk Protein Precursors --«—— - _ . l6 1. Determination of the Radio- Chemical Purity of Tour-11119-24“ and Glutamic Acid 2 C14 -..- .1 -1. 1.....- 16 2. The Production of 01 labeled Rabbit Serum Proteins and Their Fractionation —- -- 16 3. Methods of Injec oion and Withdrawal of Rabbit Blood and Milk Samples -- 18 h. Fractionation of Rabbit Milk Proteins 1 — 20 5° Dialysis of Rabbit Blood and Milk Protein Fractions --—-~——u- —— 21 6. LyOphillization of Protein Fractions -1. — 21 7. Paper Electrophoresis of Rabbit Serum, Whey, Blood a.nd Whey Protein Fractions ---------------- 22 8. Procedures for Estimation and Isolation of Free Blood Leucine and Glutamic Acid in Rabbit Blood Serum — 23 9. Procedure for the Estimation of the Percent Leucine and Glutamic Acid in Blood and Milk Protein Fractions 2h 10. Procedure for the Isolation of Glutamic Acid from Serum and Milk Protein Fractions for Estimation of the Specific Activity of Glutamic Acid 25 11. Procedure for the Estimation of Radioactive Leucine in the Blood and Milk Protein Fractigns - 26 12. Determination of the Specific Activity of C Pigtein Fractions Produced by the Injection of Labeled BaC03 — 27 -. we. . Page 13. Sample Preparation. Counting Procedure. and the Calculation of Specific Activity -111111 ----- 28 lb. Data on Urine and a Summary of Animals Used in Experiments I Through X -111119111I1_-_-----1--1- 31 B. Methods Used for In Vitro Studies of Guinea Pig Mammary Tissue - n-rmm 1_-—-~~eem~~—- 32 1. Cleaning of Glassware — --fl~m~ n-emmmmvmm-mv 32 2. Preparation of Media ~~~~~~~~~~~~~~~ -11- . 11-- 32 3. Tissue Culture Me hod - _ e‘rv~--»—~m—-— 33 b. Removal of Mammary Tissue and Preparation of Explants -uuw-....u..m .._. . -. 1. .1... ---1-..1.-..-...1 ..n_.,._.._- 314. 5. The Culture Period 1 — -- — —-_~~~~- - --- 35 6. Preparation of Stained Histological Sections of Exp3-ants and Control Tissues -111111111111112--_- 35 7. Preparation of Autoradiographs - —.~ ~--—-- - 36 8. Evaluation of the Morphological Condition of the Explants -1111“ -------------------------------------- 37 RESULTS AND DISCUSSION «an- uuuuuu 151-_111-1-1--s_--m11_ ~~—v — 38 I. Studies on the PrecuIs0Is of Rabbit Milk Proteins ~~~~~~~ 38 A. EXpeIiment l. The Inc orporat.ion of Leucine-Z C12+ into Serum and Milk Proteins by the Lactating Rabbit— 38 l. Decrease in the Specific Activity of the Extra~ cellular Free Leucine Pool -11121 ~~~~~~~~~~~~~~~~~ 38 2. Decrease in the Specific Activity of Free Blood leucine. Comparison of Results with Other Work-- 50 3. Free Blood Leucine as a Direct Precursor of Caseins. B -Lactoglobulin and oL—Lactalbumin of Milk »--— - — -— ——--- 58 4. The Relationship Between.V’~Globulins of the Blood and the Immune G1 obulins of Milk ----- « ----- 61 5. The Origin of "Blood" Serum Albumin in Rabbit Milk - — — ....... 7o 6. The Specific Activity of Leucine Incorporated into the DC - and B -Globulins of Blood ---~ ------ 71 B. Experiments 3, 3 and h. The Incorpo ation of Leucine-2—C1 and G lutamic Acid— 2- Cl into Serum and Milk Proteins by Lactating Rabbits — - 80 C. Experiments 5 and 6. Experiments Designed to Demonstrate the Transfer of BloodTYC-Globulins to Mammary Secretions of the Rabbit --- 136 D. Experiments 7 and 8. Experiments Designed to Demonstrate the Transfer of Blood Albumins to Mammary Secretions of the Rabbit — 1&8 E. EXperiments 9 and 10. EXperiments Designed to Evaluate the Potential of o(-Globulins and £9-Globulins as Precursors of Rabbit Milk Proteins -- 159 II. Page Studies of the Hormonal RequiIements of Guinea Pig Mammary Tissue In Vitro -111:1111111111a__11111111111-1-- 160 Results A. B. 1. 2. 3. 4. 5. Development of Tissue Culture Mediums -~a~awawm~~ 160 Results of EXplants Prepared from Nona Secretory Guinea Pig Mammary Tissue and Cultured in Medium I —- ~-- 164 Results of Explants Prepared from Secretory Guinea Pig Mammary Tissue and Cultured in Medium II ~ ~~e~ 1.1...... ~ --~- 169 Results of Explants Prepared from Secretory Guinea Pig Mammary Tissue and Culturgd in Medium II containi.ng DL-Leuoine-ZWC1 or DIP Glutamic Acid 2 C14 -~m~~_-~~~ — - 176 Results of Expiants Prepared from Secretory Guinea Pig Mammary TissBe and Cultured in 14 Medium.II containing Ct Y’ -Globulin or Cl Albumin -_111. — __1_ ~ - 179 Discussion 1. 2. Preliminary Studies on the Cultivation of Rat. Mouse and Rabbit Mammary Tissue IQ'VthQ. in 'Which Negative Results were Obtained —~~_ 181 DevelOpment of Mediums for the Maintenance of Secretory and Non-Secretory Guinea Pig Mammary Tissue Explants In Vit .19- ,1_. - 183 SUMMARY --_--------- — —-=~~1 , - — __ 19o BIBLIOGRAPHY ___1_11 _ — —r—— _1 -——— --— 198 APPENDIX ._1 . 11-1--11----- ........... 207 Table lo 20 3. 5. 7. 9. LIST OF TABLES The Specific Activities of Leucine from Blood and Milk Protein Fractions from Lactating Rabbit (No. X—35, luth- l7thuDays Postpartum) Injected with 45.0 no. of Leucine- Ratios of the (a) Specific Activities of Leucine from Milk Immune Globulins to the (b) Specific Activities of Leucine from Blood.Y’-Globulins from Lactating Rabbit (No. X~35, 14th-l 17th Daze Postpartum) Injected with 45. O.pc. of Leucine-Z-C —-- Ratios of the (a) Specific Activities of leucine from "Blood" Serum Albumin from Milk to the (b) Specific Activ- ities of Leucine from Blood Albumin from lactating Rabbit (No. X—35, 14th-l7tE Days Postpartum) Injected with #5. 0 no. of Leucine-Z-Cl --- Specific Activities of Leucine from Blood and Milk Protein Fractions from a Lactating Rabbit (No. X-32, 14th-l7th h Days Postpartum) Injected with #5.0 pc. of Leucine-Z-Cl -- Ratios of the (a) Specific Activities of Leucine from Milk Immune Globulins to the (b) Specific Activities of Leucine from Blood Y'—Globulins from a Lactating Rabbit (No. X-32, lath-17th Daze Postpartum) Injected with 45. 0 yo. of Leucine-Z-Cl ------------ Ratios of the (a) Specific Activities of Leucine from "Blood" Serum Albumin from Milk to the (b) Specific Activ- ities of Leucine from Blood Albumin from a Lactating Rabbit (No. X-32,.1uth-thh Days Postpartum) Injected with “5.0 no. of Leucine-Z-Cl --- Specific Activities of Glutamic Acid from Blood and Milk Protein Fractions from a Lactating Rabbit (No. X-30, lhth— 17th DayiuPostpartum) Injected with 150. 0 no. of Glutamic ACid-Z-C ——- Ratios of the (a) Specific Activities of Glutamic Acid from Milk Immune Globulins to the (b) Specific Activities of Glutamic Acid from.Blood Y'-Globulins from.a Lactating Rabbit (No. X—30, lath-17th Days Postpartum) Injected with 150.0 no. of Glutamic Acid—2-014 -.. Ratios of the (a) Specific Activities of "Blood” Serum Albumin from Milk Serum to the (b) Specific Activities of Blood Albumin from a Lactating Rabbit (No. X-BO, luth- 17th Days Postpartum) Injected.with 150‘pc. of Glutamic Acid—2-01 1“ --- Page 65 75 81 85 99 102 103 Table Page 10. Specific Activities of Glutamic Acid from Blood and Milk Protein Fractions from a Lactating Rabbit (No. X-BA, 14th- l7th Days Postpartum) Injected with 150 no. of Glutamic Acid-Z- C11“ ........ 10a ll° Ratios of the (a) Specific Activities of Glutamic Acid from Milk Immune Globulins to the (b) Specific Activities of Glutamic Acid from Blood V’-Globulins from a Lactating Rabbit (No° X-34, luth-l7th Days Postpartum) Injected with 150 pc. of Glutamic Acid_2-014 ...... 107 12. Ratios of the (a) Specific Activities of Glutamic Acid from "Blood" Serum Albumin from Milk to the (b) Specific Activities of Glutamic Acid from Blood Albumin from a Lactating Rabbit (No. X-BA, lhth-l7th Days lEostpartum) Injected with 150|pc. of Glutamic Acid-Z-Cl —— 108 13. The Specific Activities of Blood V'-01obulins and Milk Immune Globulins from a Lactating Rabbit (No. X-18,10th- th Days Postpartum) Injected with O. 31.pc. (#00 mg.) CE Labeled.Y'-Globulins _ _ ---- 137 la. The Specific Activities of Blood‘T‘-01obulins and Milk Immune Globulins from a Lactating Rabbit (No. X-l9, 10th- lBth Days Postpartum) Injected with 2.0 pc. (800 mg.) C 4 Labeled Y'-Globulins ----- 138 15° Ratios of the Specific Activities of the (a) Milk Immune Globulins to the (b) Specific Activities of Blood Y’-Globulins in a Lactating Rabbit (No. X-l8, lOth—l th Days Postpartum) Injected with O.31.pc. (400 mg.) C1 Labeled77’—Globulins —~ ------- 141 16. Ratios of the Specific Activities of the (a) Milk Immune Globulins to the (b) Specific Activities of Blood Y'—Globulins in a Lactating Rabbit (NO. X-19, lOth-g3th Days Postpartum) Injected with 2.0 pc. (800 mg.) C1 Labeled Y -Globulins — =— 1142 17. The Specific Activities of Blood Albumin and "Blood" Serum Albumin from.Milk from a Lactating Rabbit (No. X-l7, 10th- 13th Days Postpartum) Injected.with 5.16‘pc. (2. 0 gm.) 01“ Labeled Albumin 149 Table 18. 19. 20° 22. 230 2b. 25. The Specific Activities of Blood Albumin and "Blood" Serum Albumin from L11k fiom a Lactating Rabbit (Mo. X-30,10th_ 1T h Days Postpartum) Injected with 13.2‘pc. (3.0 gm.) Cl Labeled Albumin mwwue ------------------- 11 eeeeeeee ~~~ew~~~~m-~-~-u The Ratios of the (a) Specific Activities of "Blood" Serum Albumin from Milk to the (b) Specific Activities of Blood Albumin from a Lactating Rabbit (No. X-l7, 10th- 13th Days Postpartum Injec ted with 5 1o pCo (2 0 gm ) cl lit Labeled Albumin -~ume uuuuuuuuuu -- .111- -------------- - Ihe Ra ios of the (a) Specific Activities of "Blood" Serum Albumin from Milk to the Specific Activities of Blood Albumin from a Lactating Rabbit (No. X-20,1Oth713th Days Postpartum) Injected with 13 2 pc. (3 0 gm.) 014 Labeled Albumin -1- ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Composition of Medium I Developed for Maintenance of Guinea Pig Mammary Tissue In_Vjt:o ~~~~~-»-~~w~~umumnwe~_- Composition of Medium II Developed for Maintenance of AC ive Secretion of Guinea Pig Mammary Tissue In Vjtro -_- Composition of Control Medium for Guinea Pig Mammary Tissue In Vjtrg mesa—a 1n__ __ Results of Explants Prepared from Non—Secretory Guinea Pig Mammary'Tissue and Cultured in Medium I for Five Days ---- Results from Explants Prepared from Secretory Guinea Pig Mammary Tissue and Cultured in Medium II for Five Days —-- Appendix 1. 2. 3. The Yields, Specific Activities and Percent of Injected Dose of Serum Protein Fractions Isolated from Male Rabbits (X-ll and X-12) Injected with 1.50 ms. of Ba003-014 ...... A Summary of the Treatments of the Lactating Rabbits Utilized in EXperiments 1 Through lO — --_- Free Leucine Levels in the Serum of X—35 (lactating Rabbit 13th-17th Days Postpartum Injected with 11.5 mg. of DL—Leucine-Z c1”) — ___ _- Average of Ten Values with Standard Errors for the Percent of Leucine and Glutamic Acid in Rabbit Serum and Milk Proteins - _ a a _-- Page 150 151t 155 161 162 163 165 170 209 210 213 214 \t *3 The Radioactivity of Urine from Xu35 (Lactating Rabbit 13th 17th Davs Postpartum Injected with 45. 0 no. of D1» Leucine 2 C1 --eeaae---_ ................................... 215 Kn o 6. Specific Activities of Blood and Milk Protein Fractions From a Lactating Rabbit (No. X 18,10th-l3th Days Post- partum) Injected with 0.31 pc. (LOO mg.) C14 Labeled Y’-Globulins ------------------------------------------------- 216 7. Specific Activities of Blood and Milk Protein Fractions from a Lactating Rabbit (No. X—l9, 10th— 13th Days Post— partum) Injected with 2 0 uc. (800 mg ) of 01 Labeled Y's Globulins -1 ..... ~_~ee~~a ------- - ------------------------------- 218 8. Specific Activities of Blood and Milk Protein Fractions from a Lactating Rabbit (No. X 17,10th-l3th Days Post- partum) Injected with 5 .16 pc. (2.0 gm.) C14 Labeled Albumin —-a-a- ..... . _--m—-eaea-- ............. --__ 220 9. Specific Activities of Blood and Milk Protein Fractions from a Lactating Rabbit (No. CX-20, 10th-l3th Days Postu pattum) Injected with 132 (3. 0 gm.) of 01+ Labeled Albumin ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 222 10. Specific Activities of Blood and Milk Protein Fractions from a Lactating Rabbit (No. X- 2A, lOth—llth Days Post— partum) Injected with 1.63lyc. (L92 mg.) C1 Labeled O(-Globulins 1-1- ----------------------------------------------- 224 11. Specific Ac.tivities of Blood and Milk Protein Fractions From a Lactating Rabbit (No. X—27, 10th-11th Days Post- partum) Injected with 2 .37 pc. (398 mg.) of C1 Labeled p -Globulins ---------------------------------------------- 226 Figure l. 2. 3. 5. 7. 9. LIST OF FIGURES The Log of the Specific Activity (pc./mm.) vs. Time (hrs. post inj.) for Free Blood Leucine and Leucine from Casein Isolated from X~35 (Lactating Rabbit l4th-l7th Days Postu partum Injected with 45.0‘pc. of DL-Leucine~2- Cl -—-—-~ iModel for the Removal of DL-Ieucine-Z-Cl’+ from the Extra- cellular P001 to the Leucine Pool of the Reticulo- Endothelial System (RES). the Intracellular Pool (HOHin) and Plasma Protein Binding - ------ Model for the Turnover of the Extracellular Pool of Leucine Two Hours Post Injection _ _«— _ -__ The Log of the Specific Activity (pc./mm.) vs. Time (hrs. post inj.) for Free Blood Leucine and Leucine from )3- Lactoglobulin Isolated from x-35 (lactating Rabbit l4th-l7th DaZ Postpartum Injected with 45. O‘pc. of DL— Leucine-Z—Cl ) The Log of the Specific Activity (pc./mm.) vs. Time (hrs. post inj.) for Free Blood Leucine and Leucine from o(- -Lactalbumin Isolated from X-35 (Lactating Rabbit l4th— l7th Days POE) tpartum Injected with 45.0 ,uc. of DL— Leucine-Z-Cl m— =---- The Log of the Specific Activity (pc./mm.) vs. Time (hrs. post inj.) for Free Blood Leucine and Leucine from Blood Y-Globulins Isolated from L3 5 (Lactating Rabbit l4th- l7th Days Poitpartum Injected with 45. O‘pc. of DL— Leucine—Z— Cl — _-- The Log of the Specific Activity (pc./mm.) vs. Time (hrs. post inj.) for Free Blood Leucine and Leucine from Milk Immune Globulins Isolated from X-35 (Lactating Rabbit l4th-l7th Days Postpartum Injected with 45.0 pc. of DL_ Leucine-Z—C ———- The Log of the Specific Activity (pc./mm.) vs. Time (hrs. post inj.) for leucine from Blood Y-Globulins and Leucine from Milk Immune Globulins Isolated from X-35 (Lactating Rabbit l4th-l7th Days Postpartum Injected with 45.0‘pc. of Dlpleucine-Z-C = — Ratios of the Specific Activities of Leucine from Milk Immune Globulins to the Specific Activities of Leucine from Blood Y’-Globulins Isolated from X-35 (Lactating Rabbit l4th-l7th CDfiys Postpartum Injected with 45. O‘pc. of DL-Leucine-Z-C1 ------ Page 42 45 48 55 56 62 63 64 Figure 10. ll. 12. l3. l4. l5. l6. 17. The Log of the Specific Activity (pc./mm.) vs. Time (hrs. post inj.) of Free Blood leucine and Leucine from Blood Albumin Isolated from X—35 (Lactating Rabbit l4th-l7th DI4 v; Postpartum Injected with 45.0 pc. of Dlpleucine-Z- The Log of the Specific Activity (uc./mm.) vs. Time (hrs. post inj.) of Free Blood Leucine and Leucine from "Blood" Serum Albumin from Milk Isolated from X-35 (Lactating Rabbit l4th-l7th Days Pos partum Injected with 45. O‘pc. of DL—Leucine—Z- C 4) _—_ _ __ —- The Log of the Specific Activity (uc./mm.) vs. Time (hrs. post inj.) of Leucine from Blood Albumin and Leucine from Blood Albumin from M1 1k Isolated from X- 35 (Lactating Rabbit l4th-17th Days Postpartum Injected with 45.0 no. of Dlpleucine-Z C14) —— Ratios of the Specific Activities of Leucine from "Blood" Serum Albumin from Milk to the Leucine from Blood Albumin Isolated from X-35 (Lactating Rabbit l4th-l7th Days Post— partum Injected with 45.0 uc. of Dlpleucine»2_C1“) ------- The Log of the Specific Activity (pc./mm.) vs. Time (hrs. post injo) of Free Blood Leucine and Leucine from Blood CK—Globulins Isolated from X—35 (Lactating Rabbit l4th- l7tEuDays Postpartum Injected with 45.0 pc. of DLpLeucine- 2- C ) — — _ ----- The Log of Specific Activity (pc./mm.) vs. Time (hrs. post inj.) of Free Blood Leucine and Leucine from Blood B-Globulins Isolated from X— 35 (lactating Rabbit 14th- l7th Days Poatpartum Injected with 45. O‘pc. of DL— Ieucine-Z- Cl _ —-------- The Log of Specific Activity (uc./mm.) vs. Time (hrs. post inj.) for Free Blood Leucine and Leucine from Casein Isolated from X-32 (Lactating Rabbit l4th-l7th Days Post- partum Injected with 45. O‘pc. of DL-Leucine- 2- Cl ------ The Log of Specific Activity (pc./mm.) vs. Time (hrs. post inj.) for Free Blood Leucine and Leucine from q-Iactoglobulin Isolated from X-32 (Lactating Rabbit l4th—l7th Days Postpartum Injected with 45.0 no. of DIP Leucine-2-C1—--— Page 72 73 74 76 77 78 86 87 Figure 18. 19. 20. 21. 22. 23. 24. 25. Page The Log of the Specific Activities Quc./mm.) vs. Time (hrs. post inj.) for Free Blood Leucine and Leucine from c(eLactalbumin Isolated from X—32 (Lactating Rabbit 14th» l7thuDays Postpartum Injected with 45. 0 pc. of DL~Leucine- Cl ) _ g 1. _ _ _l_- 88 The Log of the Specific Activities (pc./mm.) vs. Time (hrs. post inj.) for Free Blood Leucine and Leucine from Blood Y’wGlobulins Isolated from X-32 (Lac ating Rabbit 14thml7th Days Postpartum Injected with 45. O uc. of DL- Leucine-2.0i m — -- 89 The Log of the Specific Activities (pc./mm.) vs. Time (hrs. post inj. ) for Free Blood Leucine and Leucine from Milk Immune Globul.ins Isolated from X_32 (Lactating Rabbit l4th-l7th Days Postpartum Injected with 45. O‘pc. of DL— Leucine-2e C1 -111 rrrrr --- ————— 90 The Log of the Specific ActiVities (pc./mm.) vs. Time (hrs. post inj.) for Leucine from Blood Y'-Globulins and Milk Immune Globulins Isolated from X-32 (Lactating Rabbit l4th-l7tR Days Postpartum Injected with 45. 0 pc. DLeLeucine-Z-C1un- 1-- _1_ _~-- 91 Ratios of the Specific Activities of leucine from Milk Immune Globulins to the Specific Activities of Leucine from Blood‘r'-Globulins Isolated from X-32 (Lactating Rabbit l4th_l7th D ys Postpartum Injected with 45.0 pc. of DL-LeucineuL c1 ) 92 The Log of Specific Activity'(pc./mm.) vs. Time (hrs. post inj.) for Free Blood Leucine and Leucine from Blood Albumin Isolated from X—32 (Lactating Rabbit l4th-l7th Digs Postpartum Injected with 45.0 pc. of DLuLeucine-Z- C --- 93 The Log of Specific Activity (pc./mm.) vs. Time (hrs. post inj.) for Free Blood leucine and Leucine from "Blood" Serum.Albmmin from Milk Isolated from X-32 (Lactating Rabbit l4th-17th “Days Postpartum Injected with 45.0 pc.o of DL—Leucine-Z- Cl 4) — —— 94 The Log of Specific Activity (pm/mm.) vs. Time (hrs. post inj.) for leucine from Blood Albumin and "Blood" Serum Albumin from X-32 (Lactating Rabbit l4th-l7th Days Postpartum Injected with 45. 0 pc. of D1'.--Leucine--2-C1 ) .. 95 Figure 26. 27. 28. 29. 30. 31. 32. 33. Ratios of the Specific Activities of Leucine from "Blood" Serum Albumin from Milk to the Specific Activities of Leucine from Blood Albumin Isolated from X-32 (Lactating Rabbit l4th~l7th Days Postpartum Injected with 45. O‘pc. of DIpIeucine-Z- C 1L’) —— -1,_ __-- The Log of Specific Activity'(pc./mm.) vs. Time (hrs. post injo) for Free Blood Leucine and Leucine from Blood c2 Globulins Isolated from x~32 (Lactating Rabbit l4th— l7t§uDays Postpartum Injected with 45. 0 pc. of DL—Leucine- 2 C ) u—_ _ The log of Specific Activity (uc./mm.) vs. Time (hrs. post injo) for Free Blood Leucine and Leucine from £3~Globulins Isolated from X-32 (Lactating Rabbit l4th— l7thuDays Postpartum Injected with 45. O uc. of DL—Leucine- ) . The Log of the Specific Activity (uc./mm.) vs. Time (hrs. post inj.) for Free Blood Glutamic Acid and Glutamic Acid from Casein Isolated from X-BO (Lactating Rabbit l4th- l7th Days Postpartfim Injected with 150. O‘pc. of DL~ Glutamic Acid—2_C*) «mm- The Log of the Specific Activity'(pc./mm.) vs. Time (hrs. post inj.) for Free Blood Glutamic Acid and Glutamic Acid from B -Lactoglobulin Isolated from X-BO (lactating Rabbit l4th-l7th Days Postpartum Injected with 150. O‘uc. of DLeGlutamic Acid_2 c j “=_-—. 1.11. -- The log of Specific Activity (pc./mm.) vs. Time (hrs. post inj. ) for Free Blood Glutamic Acid and Glutamic Acid fitm10<-Iactalbumin Isolated from X-BO (Lactating Rabbit l4th-l7th Days Postpartum Injected with 150. O‘pc. of DL— Glutamic Acid-2-c14) _ The Log of Specific Activity (pc./mm.) vs. Time (hrs. post inj.) for Free Blood Glutamic Acid and Glutamic Acid from Blood'Y'-Globulins Isolated from X-BO (lactating Rabbit l4th-l7th Days Postpartum Injected with 150.0‘pc. of DL-Glutamic Acid—2-014) The Log of Specific Activity (pc./mm.) vs. Time (hrs. post inj.) for Free Blood Glutamic Acid and Glutamic Acid from Milk Immune Globulins Isolated from X—3O Lactating Rabbit l4th-l7th Days Postpartum Injected with 150.0 no. of DIPGlutamic Acid-2-014 ) Page 96 97 98 109 110 111 112 113 Figure Page 34. The Log of the Specific Activity (pc./mm.) of Glutamic Acid from Blood Y'-Globulins and Milk Immune Globulins Isolated from X-30 (Lactating Rabbit l4th-17th Days Pogtpartum Injected with 150.0'pc. DL—Glutamic Acid-2- cl ) --- 114 35. Ratios of the Specific Activities of Glutamic Acid from Milk Immune Globulins to the Specific Activities of “Glutamic Acid from Blood Y’-Globulins from X-30 Lactating Rabbit 14th-17th Days lEostpartum Injected with 150 pc. of DL-Glutamic Acid-Z-Cl —- 115 36. The Log of the Specific Activity (pc./mm.) vs. Time (hrs. post inj.) for Free Blood Glutamic Acid and Glutamic Acid from Blood Albumin Isolated from X-30 (Lactating Rabbit 14th-17th Days Postpartum.Injected with 150. O‘pc. of Dlp Glutamic Acid—2-014) 116 37. The Log of the Specific Activity (uc./mm.) vs. Time (hrs. post inj.) for Free Blood Glutamic Acid and Glutamic Acid from "Blood" Serum Albumin from Milk Isolated from X—30 (Lactating Rabbit 14th-17th Days Pastpartum Injected with 150.0 pc. of DL-Glutamic Acid-2- C:L --------- 117 38. The Log of the Specific Activity'(uc./mm.) vs. Time (hrs. post inj.) for Glutamic Acid from Blood Albumin and “Blood" Serum Albumin from Milk Isolated from X-30 (lactating Rabbit 14th-17th Day Postpartum Injected with 150.0 pc. DlpGlutamic Acid-2-C 118 39. Ratios of Specific Activities of Glutamic Acid from "Blood" Serum Albumin from Milk to the Specific Activities of Glutamic Acid from Blood Albumin Isolated from X-30 (Lactating Rabbit 14th-l7th Days Pgstpartum Injected with 150.0‘pc. of DL—Glutamic Acid-2-01 ) 119 no. The log of the Specific Activity'(pc./mm.) vs. Time (hrs. post inj.) for Free Blood Glutamic Acid and Glutamic Acid from Blood.O(-Globulins Isolated from X-30 (Lactating Rabbit 14th-l7th Days Postpartum Injected with 150. 0 pc. of DL—Glutamic Acid-Z-C1 4) 120 41. The Log of the Specific Activity'(uc./mm.) vs. Time (hrs. post inj.) for Free Blood Glutamic Acid and Glutamic Acid from Blood fl -Globulins Isolated from X-30 (lactating Rabbit 14th-17th Days uPostpartum Injected with 150. 0 pc. DL-Glutamic Acid-Z-Cl 4) -- 121 Figure 42. 43. 45. 46. 47. 48. 49. The Log of Specific Activity (uc./mm.) vs. Time (hrs. post inj.) for Free Blood Glutamic Acid and Glutamic Acid from Casein Isolated from X-34 (Lactating Rabbit 14th- 17th Days Postpartum Injected with 150.0 pc. of DL- Glutamic Acid_2-cl“) u ....... The Log of the Specific Activity'(uc./mm.) vs. Time (hrs. post inj.) for Free Blood Glutamic Acid and Glutamic Acid from B -lactoglobulin Isolated from X—34 (lactating Rabbit 14th~l7th Days Postpartum.Injected with 150. inc. of DLuGlutamic Acid- 2 014) ~_ —-- The Log of the Specific Activity (uc./mm.) vs. Time (hrs. post inj.) for Free Blood Glutamic Acid and Glutamic Acid r:m1&¢- Lactalbumin Isolated from X-34 (Lactating Rabbit 14th 17th Days Postpartum Injected with 150. 0 pc. of DL— Glutamic Acid 2 cl ) __. ..... ---- The Log of the Specific Activity (pc./mm.) vs. Time (hrs. post inj.) for Free Blood Glutamic Acid and Glutamic Acid from Blood Y’—Globulins Isolated from X- 34 (Lactating Rabbit 14th—17th Days Pogtpartum Injected with 150. 0 pc. of DLPGlutamic Acid- 2 C1 ............. The Log of the Specific Activity (uc./mm.) vs. Time (hrs. post inj.) for Free Blood Glutamic Acid and Glutamic Acid from Milk Immune Globulins Isolated from X-34 (Lactating Rabbit 14th—l7th Days Postpartum Injected with 150. O‘uc. of Glutamic Acid- 2— C14) —-- _ _ The Log of the Specific Activity (uc./mm.) vs. Time (hrs. post inj.) for Glutamic Acid from Blood.T’-Globulins and Milk Immune Globulins Isolated from X-34 (Lactating Rabbit l4th-17th Days Postpartum Injected with 150. 0 pc. of Glutamic Acid-Z— C 1I") -— Ratios of the Specific Activities of Glutamic Acid from Milk Immune Globulins to the Specific Activities of Glutamic Acid from‘Y‘-Globu1ins Isolated from X-34 (Lactating Rabbit 14th-l7th Days Pastpartum.lnjected with 150.0 pc. of DLPGlutamic Acid-Z-Cl ) The Log of the Specific Activity'(pc./mm.) vs. Time (hrs. post inj.) for Free Blood Glutamic Acid and Glutamic Acid from Blood Albumin Isolated from X-34 (Lactating Rabbit l4th-l7th Days Postpartum Injected with 150.0‘pc. of DL— Glutamic Acid_2—014) Page 122 123 124 125 126 127 128 129 Figure 50. 53. 54. 55- 56. 57. The Log of the Specific Activity (pc./mm.) vs. Time (hrs. post inj. ) for Free Blood Glutamic Acid and Glutamic Acid from "Blood" Serum Albumin from M114 Isolated from X— 34 (lactating Rabbit l4Th- l7ih Days Postpartum Injected with Page 150. 0 pc. of DIPClutamic Acid- 2- Cl“) -—~~- ~~~~~~~~~ - --------------- 130 The Log of the Specific Activity (pc./mm.) vs. Time (hrs. post inj.) for Glutamic Acid from Blood Albumin and Glutamic Acid from "Blood" Serum Albumin from Milk Isolated from X-34 (lactating Rabbit l4thol7ih Days Post- partum Injected with 150.0 pc. of DL—Glutamic Acid-2_Clu)— Ratios of the Specific Activities of Glutamic Acid from "Blood" Serum Albumin from Milk to the Specific Activi~ ties of Glutamic Acid from Ilood Albumin Isolated from X- 34 (Lac tating Rabbit l4 thel7th Days Postpartum Injected with 150 o no . of DL GluLimic Acid 2 c1 ) -Um-i-l- cm---- The Log of the Specific Activity (pc./mm.) vs. Time (hrs. post inj.) for Free Blood Glutamic Acid and Glutamic Acid from Blood‘kf-Clobulins I.solated ft om X- 34 (Lactating Rabbit 14thJ 7th Dajrs Postpartum Injected with 150. 0 pc. of Glutamic Acid 2 C ”) --—-~m~~ —~.~--~~---~--_—-—------ The Log of Specific AC tivity (pc. /mm.) vs. Time (hrs. post inj.) for Free Blood Clu+amio Acid and Glutamic Acid from Blood fi3-Globulins Isolated flom X—34 (lactating Rabbit l4th-l7th Days Postpartum Injected with 150.01pc. of DleGluiamic Acid—2-c1”) —- _ _-___-___ The Log of the Specific Activity (pc./mg. x 104) vs. Time (hrs. post inj.) for Blood'Y’-Globulins and Immune Globulins from Milk Isolated from X-18 (Lactating Rabbit 10th-13th Days Postpartum Injected with 0.13 pc. C Labeled Y'-G10bu1in) ---~- ----------------------------- ~---- The Log of the Specific Activity (uc./mg. x 104) vs. Time (hrs. post inj.) for BloodYY’—Globulin and Immune Globulins from Milk Isolated from X-19 (Lactating Rabbit lOth—l3th Days Postpartum Injected with 2.0 pc. of Cl Labeled‘Y'-Globulins) — — ---------- Ratios of the Specific Activities of Immune Globulins from Milk to the Specific Activities of Y’—Globulins in Blood Isolated from X— 18 (Lactating Rabbit lOth- 13th Days Postpartum Injected with o. 3l.pc. of 014 Labeled V—Globulins) --- —— _ — --------------------- 131 132 133 134 139 Figure 58° 59. 60° 61. 62. 65. 66. 67. Ratios of the Specific Activities of Immune Globulins from Milk to the Specific Activities of Y‘—Globulins in Blood Isolated from X-l9 (Lactating Rabbit lOth-lBth Days Post- partum Injected with zoo‘uo° of c1 Labeled.Y'-Globulins)- The Log of the Specific Activity (peg/mg. X lOu) vso Time (hrs. post injo) for Blood Albumin and "Blood" Serum Albumin from Milk Isolated from X—l7 (Lactating Rabbit 10th.l3th Days Postpartum Injected with 5°16‘pc. of 014 Labeled Albumin) —~=— — _ ------ The Log of the Specific Activity (pm/mgo x 10“) vs. Time (hrs° post inj.) for Blood Albumin and "Blood" Serum Albumin from Milk Isolated from X-ZO (lactating Rabbit lOth-lBth Days Postpartum Injected with 13.2 no. of 014 Labeled Albumin) ——~~ -— — -- Ratios of the Specific Activities of "Blood" Serum Albumin from Milk to the Specific Activities of Blood Albumin from X—l7 (Lactating Rabbit 10th-l3th Days Post- partum Injected with 5.16 pen of cl“ Labeled Albumin) ..... Ratios of the Specific Activities of "Blood" Serum Albumin from Milk to the Specific Activities of Blood Albumin from X-ZO (Lactating Rabbit lOth-lBth Days Post- partum Injected with 13.2 p00 of cl“ Labeled Albumin) ---- Photomicrograph (lBOX) of a Non-Secretory Control Guinea Pig Mammary Tissue Section Stained with Iron Hematoxylin and Eosin -—- _. ~—— -~~ ~ ~ — ------ Photomicrograph (lBOX) of a Section of an Explant Prepared from the Same Animal as the Control Tissue (Figure 63) and Cultured in Medium I for Five Days ----- Photomicrograph (lBOX) of a Secretory Control Mammary Tissue Taken from a Lactating Guinea Pig. Stained with Iron Hematoxylin and Eosin =— ----- Photomicrograph (lBOX) of a Section of an Explant Taken from the Same Tissue Shown in Figure 65 and Cultured for Five Days in Medium II --------------------------------- Photomicrograph (lBOX) of a Section of an Explant Taken from the Same Tissue Shown in Figure 65 and Cultured for Five Days in the Control Medium ---—— — —— Page 144 151 152 156 157 168 169 173 174 175 Figure 68. 69. 70. Photomicrograph of an Autoradiograph Prepared from an Explant Cultured for Five Days in Medium II Containing O. 2'pc. /m1. of DL—Glutamic Acid-2-C14- ~ -_--———— Photomicrograph of an Autoradiograph Prepared from an Explant Cultured for Five Days in Medium II Containing 0.2 uc./ml. of DL—Ieucine-Z-Clu _-.. Photomicrograph of an Autoradiograph Prepared from an Explant Cultured £or Five Days in Medium II Containing 0.1 pc./m1. of cl Labeled r-Globulin .................. Appendix 1. Photographs of Paper Chromatograms of DIPIeucine-Z-Clu and DIFGlutamic Acid- 2— C1 with Recordings of Radio- activity --~m-~—u ----------------------------------------- Photograph of Paper Electrophoresis Patterns of Rabbit Serum and Protein Fractions Isolated from the Serum ----- Photograph of Paper ElectrOphoresis Patterns of Rabbit Whey and Protein Fractions Isolated from the Whey ------- The Log of Specific Activity (pc./mg. x 105) vs. Time (hrs. post inj.) for Blood Oc-Globulins, Blood )f-Globulins and Immune Globulins from Milk Isolated from X—24 (Lactating Rabbit lOth-llth Days Postpartum Injected with l. 63 no. of cl“ Labeled o¢-Globulins _____ The Log of Specific Activity (uc./mg. x 105) vs. Time (hrs. post inj.) for Blood B-Globulins, Blood ‘YLGlobulins and Immune Globulins from Milk Isolated from X—27 (Lactating Rab 't lOth—llth Days Postpartum Injected with 2.37 no. of 01 labeled p -Globulins — _-_=-— Page 177 178 180 208 211 212 228 Dedicated to my lovely. patient wife Marian and son Steven I~~I as, h o.- . F- '.A.. ACKNOWLEDGEMENT The author wishes to express his sincere gratitude and deepest appreciation to Dr. Joseph Meites, professor of the Department of Physiology and Pharmacology, for his continual encouragement and support during the course of this investigation and for his generous assistance and constructive criticism during the preparation of this thesis. He also wishes to express his appreciation to Dr. B. V. Alfredson, head of the Department of Physiology and Pharmacology, for providing facilities and laboratory space to carry on these experiments. Sincere thanks are due Drs. J. E. Nellor and P. O. Fromm, Department of Physiology; Dr. R. IM. Grimes, formerly of the Department of Agricultural Chemistry; Drs. R. 'U. Byerrum and H. A. Lillevik, Department of Chemistry; and Dr. J. R. ‘Brunner of the Department of Dairy for providing facilities and apparatus used during the course of these investigations. Special thanks are due Dr. Esther M. Smith of the Department of Anatomy for invaluable advice and assistance on tissue culture techniques. The author also wishes to express his sincere thanks to Dr. L. F. 'Wolterink, Department of Physiology, for his help and advice during the course of this investigation and for his assistance in the preparation of this thesis. Also, special thanks are due Dr. H. G. Hansen, head of the Department of Agricultural Chemistry, for use of the ;?-Liquid Tricarb Scintillation Spectrometer and for his assistance in preparation of this thesis. The author also wishes to thank Mr. M. R. Swab for his help in the care of experimental animals, and to express his Special appreciation to Mr. C. S. Nicoll for his suggestion to try guinea pig mammary tissue for culture studies. 4 u no ‘5 a. ‘ 'vw- The author wishes to thank the U. 8. Public Health Service, National Institutes of Health, for a Pre-Doctoral Fellowship during the last two years of this work. He is also obligated to the Department of Physiology and Pharmacology for a Teaching Assistantship during the first year of thiS‘work. Special thanks are also due to the Institutional Research Grant Committee for a grant of $500 towards the purchase of radioactive amino acids used in the course of these investigations. Thanks are also due The Endocrinology Study Section, National Institutes of Health, for pro- viding prolactin; to Dr. L. Michaud of Merck, Sharp and Dohme, for supplying hydrocortisone; and Dr. R. Kraay of Eli Lilley, for supplying the insulin used in these investigations. The writer is also indebted to the Michigan Agricultural EXperiment Station and the National Institutes of Health for providing financial support to Dr. Joseph Meites which helped the author to carry out this work. INTRODUCTION The mosynthesis of proteins is an extremely complex process. The synthesis of native proteins in mm has never been accomplished, al- though brilliant investigations have led to the synthesis of peptides such as those found in insulin. The problem of protein synthesis has fascinated scientists for many years and has stimulated a tremendous amount of research in this area. The mammary gland in full lactation is one of the most active syn- thesizers of proteins in the animal body. Milk is essential for the nutrition of the newborn and is one of the most important sources of high quality protein, readily available as a nutrient for human nutri- tion. Since the mammary gland lies outside of the bocb' cavity, is readily accessible, and has a rapid rate of protein synthesis, it is an excellent organ for the study of the precursors of proteins; The precursors of proteins in the milk have been studied by the following techniques: (1) arterio-venous differences (2) perfusion mtperiments (3) comparisons of electmphoretic mobilities and amino acid composition of blood and milk proteins (it) most recently, by isotOpe techniques. The data from these experiments have provided a great deal of information of a qualitative nature concerning the precursors of milk Pmteins. However, there is a decided lack of quantitative information, concerning the precursors of milk protein. Also, a considerable amount 01’ the literature on the precursors is conflicting. This is especially true or the early work on arterio-venous studies. This thesis is mainly concerned with an attempt to throw additional light upon the problem of milk protein precursors in the rabbit by pro- Viding 3 (1) quantitative information on the role of free blood leucine and glutamic acid as precursors of milk proteins (2) quantitative infor- mation on the role of blood.wrtglobulins as the source of immune globu- line in rabbit milk and (3) quantitative information on the source of "blood“ serum albumin in rabbit milk. Radioactive leucine and glutamic acid were chosen for these investigations because of their relatively high concentration in milk proteins. Also, leucine is an essential amino acid and glutamic acid is an extremely labile non-essential amino acid. The cost of radioactive amino acids is extremely high. Therefore, a decision had to be made concerning the animal in which the investiga- tion was to be carried out. The decision was primarily'whether to give one large dose to one larger animal, thus obtaining one set of large samples to work with, or to give a number of small doses to a small animal such as the rabbit and work with small samples. Statistically, there are disadvantages to either choice. The latter choice was made for these investigations. . There is very little information available on the role of serum proteins as precursors of milk proteins. For this reason, it was felt that these proteins should be evaluated as a possible source of milk jpmpteins. An attempt was made to evaluate the role of blood protein fractions by labeling them with c14 and injecting the fractions into 1Lactating rabbits with the subsequent isolation of the milk proteins. Recently, attention has turned to the in|yitzg cultivation of nuhmmarytissue. It was felt that this would be a good tool for the in- veatigation of the uptake ‘of amino acids and proteins by the luauumarygland, since the gland is removed from the influences of other “beans and the animal body in general. Thus, a large number of variables .i..:.~.. .i... r" f I! -0" '0 a can be controlled to a great extent. EXperiments were therefore con- ducted to develop satisfactory media for the maintenance of guinea pig mammary explants in 111m. It is hoped that the results of these experiments have helped to clarify the role of certain blood precursors in the synthesis of rabbit milk proteins and the in 213:9 hormonal requirements of guinea pig mammary tissue. However, it is felt that additional experiments must be done before these problems can be completely resolved. REVIEW OF LITERATURE I. The Biosynthesis of Milk Proteins A. Techniques Utilized for the Study of Milk Protein Precursors and the Results Obtained It is necessary to clarify the nomenclature of the various milk proteins referred to in this review of the literature on the precursors of milk proteins, since the same terminology is not employed by all workers. Jenness et_al. (1956) have clarified the rather confusing nomenclature of the milk proteins which exists in the earlier literature. They have defined the milk proteins of the cow in the following manner: 1) Caseins: 80% of milk protein, molecular weight of 24,100, isoelectric point 4.7, mobility at pH 8.6 is -6.7. 2) B-Lactoglobulin: 7-1276 of milk protein, molecular weight of 35,500, isoelectric point 5.18, mobility at pH 8.6 is -5.1. 3) oo—Lactalbumln: 2-5% of milk protein, molecular weight of 15,100, isoelectric point h.1-4.8, mobility at pH 8.6 is -h.2. 4) "Blood" Serum.Albumin: o.7-1.3% of milk protein, molecular weight of 65,000, isoelectric point 1+.7, mobility at pH 8.6 is -6.7. 5) Immune Globulins (composed of two fractions): a) Euglobulins: 0.8.1.7% of milk protein, molecular weight of 180,000, isoelectric point of 6.0, mobility at pH 8.6 is -l.8. b) Pseudoglobulins: 0.6-1.4% of milk protein, molecular weight of 180,000, isoelectric point 5.6, mobility at pH 8.6 is -2.0-2.22. This terminology will be strictly adhered to throughout the text. 1. aanhaduadBanauLlhJIhasnass_in4Uailkuxsauaaiians_af_Amina .Aehiisnxilhsusuxmunfihlaod It is obvious that the secretory products of the mammary gland are °Piginally derived from the blood passing through this gland. The basic problem is--what are the components of the blood which are the direct pre cursors of the milk proteins? Folley (1956) has stated that there are four main possibilities to consider in respect to the mechanism in- volved in the biosynthesis of milk proteins. Milk protein may be syn- thesized (l) entirely from the amino acids of the circulating blood (2) partly from the preceding and partly from amino acids arising from the degradation of blood plasma protein in the mammary gland (3) from blood plasma proteins by rearrangement of peptide chains inVOlving transpeptidation reactions and (a) they may be derived only partly from the latter and partly from blood amino acids. Arterio-venous studies can provide useful information about the uptake of a particular blood constituent by an organ. Since the mammary gland is outside of the body cavity, it is relatively easy to study the uptake of substances by the mammary gland from the blood by the arterio- venous technique. Cary (1920) was the first to study the uptake of amino acids by the mammary gland. He took blood samples simultaneously from the jugular and mammary (abdominal subcutaneous) veins of milking cows and determined the free amino nitrogen in the blood samples. He fbund that the blood from the mammary veins contained 16-34% less amino acid nitrogen than the jugular blood. Blood samples from dry cows gave values of -3 and 5% for amino acid differences and thus no uptake by the mammary gland. From these data he concluded that the amino acids removed from the blood by the mammary gland are sufficient to account for the pro- teins of milk and that they were undoubtedly the precursors of these milk constituents. The work of Cary (1920) was repeated by Blackwood (1932).. She took true arterial blood samples instead of jugular blood samples. Her arterio-venous differences for free amino nitrogen were between 8 and 15% on u lactating cows and 2 and -6% on 4 dry cows. This work also demonstrated uptake of amino acids by the secretory mammary gland. Graham (1937) attempted to compare the quantity of free amino ni- trogen absorbed from the blood by the udder of the lactating goat during the production of a given quantity of milk. He measured the blood flow through the udder with a flow meter in the mammary vein and the arterio- venous difference of free amino nitrogen was determined. The two goats were milked dry just prior to and at the conclusion of the experiment. His data indicated that all of the amino nitrogen secreted in the form of milk protein could not be accounted for by the free amino acids ab- sorbed from the blood by the mammary gland. Studies with the arterio- venous technique by Lintzel (1934), Shaw and Petersen (1938), and Reineke £3.31n (1939) have led to the same conclusion as Graham (1937) that the Uptake of amino acids by the mammary gland is not sufficient to account for all of the amino acids secreted in the form of milk proteins. Shaw and Petersen (l939a,b) stated that the amino acid uptake could not account for more than 40 to 50% of the casein nitrogen of milk. It was principally because of the general acceptance that free tflood amino acids could not account for all of the milk protein that attention was directed to the possibility of plasma proteins as milk protein precursors. Reineke £3.31, (1941) carried out arterio-venous studies which led them to the conclusion that some fraction of the plasma protein was utilized by the udder for the formation of milk pro- tein. They postulated that this fraction was probably a globulin fraction containing proteinpbound carbohydrate. Graham.§t,a1. (1938) also reported that a globulin of the blood was taken up by arterio- venous studies and utilized by the mammary gland. More recently, Nikitin (1949) has done arterio-venous studies and has reported that not more than 45% of the amino nitrogen of milk proteins comes from.free amino nitrogen in the blood. His work is in general agreement with the earlier‘work. The arterio-venous technique is subject to a number of errors which may lead to erroneous conclusions. Folley (l9h0, 1949) has critically reviewed the work done on the arterio-venous technique as a tool for measuring the blood precursors of milk constituents. In order to quan- titate data of this nature, it is necessary to determine the volume of blood that has passed through the udder during the time that the milk is being synthesized. It is relatively easy to determine the amount of milk synthesized in a given period of time. However, the flow of blood is subject to large variations. There are two methods used to determine the blood flow through the udder. Graham at 3.1. (1938) measured the blood flow directly with a flow meter and found that 150-250 volumes of blood passed through the goats udder for each volume of milk produced. The other method is based on the arterio-venous difference of calcium or phosphorus and the blood volume calculated from this value. The cal- culated values reported in the literature vary from 387 to 563. Folley (l9k9) believes the true value to be in the range of #00-600 volumes of blood to one volume of milk. ‘Values based on calcium uptake would be in error since all of the calcium.would not be secreted via the milk. Some would be returned via the lymph. Shaw and Petersen (1940) have reported that the flow of lymph from the mammary gland of the cow is considerable. Some calcium would undoubtedly go with it and cause a low blood to milk volume ratio by this method of calculation. The arterio-venous differences reported in the literature represent uptakes of averaged values over the relatively short periods of time required to collect the blood samples, and thus do not really represent the aver- age value during the entire period of the experiment. The secretion ‘ ”0" arrival I :wu I0- '0 N}. . L5“ a In A I h: *- rate of milk is determined over a period of hours, during which the rates of blood flow and milk secretion may independently undergo con- siderable variation. There is the possibility of systemic variations in the uptake of certain milk constituents from one milking to the next. This has been reported by Shaw and Petersen (19140). This could cause a very large error in arterio-venous studies. The arterio-venous differences are also subject to uncertainties arising from the possibility of their loss via the lymph and also from random or systematic changes in their rate of uptake as stated by Graham at al. (1936). Shaw and Petersen (1939) observed appreciable blood volume changes when the subject was disturbed by blood sampling procedures. This appears to be due to vasomotor effects. In order to minimize vasomotor disturbances during blood sampling, Reineke at al. (1941a) proposed the use of nembutal to anaesthetize the animal. They reported that anaes- thetized goats continued to secrete milk of normal composition at an unchanged rate. Shaw (19146) has also reported this to be true in cows anaesthetized with nembutal. Barry (1958) has strongly criticized the arterio-venous technique. However, Sheldon-Peters and Barry (1956) published results obtained by the arterio-venous technique in which they state that all of the essen- tial amino acids in casein were derived directly from the blood. Folley (19’49) states, that in view of the considerations discussed above, it Seems justifiable to conclude that quantitative balance experiments con- ducted on the mammary gland by the arterio—venous technique have very lill-tle validity; the arterio-venous technique in its present form is cathable of giving qualitative results only. LWWW The first perfusion study was done by Foa (1912) on an isolated sheep's udder. Petersen.e1‘al. (1939, 1941) developed a practical appa- ratus for the perfusion of the bovine udder which eliminated many of the difficulties encountered in the arterio-venous technique. These advan- tages are that the general disturbances to the animal are eliminated and that the actual flow of blood and lymph may be measured at all stages of the experiment. Also, the metabolites can be added to the perfusate and their fate can be studied. This technique can also be used to detect precursors which are taken up by the mammary gland in small amounts, since the blood can be recirculated to increase the con- centration.difference. Petersen et 31. stated that the gland functioned normally as evidenced by the disappearance of metabolites and normal uniform R.Q. values. Peeters and Massart (l9h7, 1952) also developed a perfusion technique for the udder independently of Petersen (1941). The major disadvantage to this procedure is the difficulty of main- taining the Optional levels of metabolites and hormones as pointed out by Reineke et_al. (19h1). It appears that the maintenance of the prOper pH and the accumulation of metabolic waste products might also be a problem. Bouckaert et_al. (1953) have utilized the perfusion technique on the isolated udder of the cow to study the uptake of free amino acids. The perfused blood was passed through the isolated udder a total of 10 times and they found a significant decrease in 10 of the free amino acids of the blood. The uptakes were usually most marked in those amino acids which are found in high concentrations in casein. Casein hydrolysate 'was added to the blood perfused through one-half of the udder. The up- take in this half was higher than in the half that was perfused with 10 blood that had no casein hydrolysate added. The work of Bouckaert et a1, (1953) tends to indicate that mammary tissue selectively takes up the amino acids which are present in casein in the greatest concentration. Recently, Peeters at al. (1957) have carried out a perfusion experiment on a lactating cow's udder with the addition of 0.5 no. of cysteine-S35 added to the perfusion blood. Their results are very interesting since they found twice as much activity in the whey proteins as in casein or plasma proteins at the end of the perfusion. Lauryssens et_al. (1957) and Peeters et a1. (1957) have perfused the cow udder with prOpionate-ClLL and glucose-Cl“. They found that the milk proteins were active when propionate-Clu'was added to the perfusion blood. Barry (l958)states in his review that McNaught and Folley (1958) have been able to demonstrate the uptake of radioactive amino acids by incubating mammary tissue slices. Also, Peeters gt,al, (1957) incubated bovine mammary tissue in.yitrg and found that the milk proteins were very slightly labeled. Tissue slices provide a useful technique for the study of precursors. However, one difficulty with this procedure is the estimation of the dry weight of the tissue, since variable amounts of milk will be retained in the tissue. This problem.has been discussed by'Folley'and Greenbaum (1947). The information obtained by perfusion and tissue slice techniques indicates that the mammary gland does take up and utilize free amino acids for the synthesis of milk proteins. The data provided by this work is of a qualitative rather than a quantitative nature. 1W .Milk_E:eieina The first application of labeled amino acids to the problem of milk protein precursors was by Campbell and Work (1952). They injected 11 valine, lysine, and glycine labeled with Cl)+ into a lactating rabbit and were able to show qualitatively that the greatest activity was present in casein and fngactoglobulin. Barry (1952) carried out simi- lar experiments on the lactating goat. He injected lysine and tyrosine intravenously and found high levels of activity in the casein. Askonas gt 31. (1954) injected radioactive glycine, valine, lysine and methio- nine into a lactating goat and found high Specific activities of the amino acids in the caseins and #9-1actoglobulin isolated from the milk. Askonas at al. (1955) injected cl” labeled amino acids into a goat. They subsequently isolated the caseins and /§Llactoglobulin from the animal's milk and subjected these protein fractions to partial hydrolye sis. They separated specific peptides by column chromatography. The specific activities of these peptides were very similar. Their work indicates that casein and fl9-1actoglobulin were synthesized from free amino acids and not peptides, since the labeling in the protein chain was uniform. Barry (1956) injected labeled glutamic acid into a lacta- ting goat. He reported that 50% of glutamic acid in the casein came directly from the free glutamic acid in the blood. Larson and Gillespie (1957) injected a large dose of cl“ labeled bicarbonate into a lactating cow. A very careful analysis of the milk proteins indicated a high level of activity in CY- and /9-casein, fl-lactoglo‘mlin and a’-lactalbumin. The activity in these protein fractions was about equal, which suggested that they were all derived from the same amino acid pool. They reported low activities in the 'kzcasein, immune globulin fraction and "blood" serum albumin of the milk which indicated that their precursors were different. Larson and Gillespie (1957) Suggested that the protein fractions of high specific activity were derived directly from the free blood amino acids. 12 Kleiber at 51. (1952) found the same type of labeling when they injected labeled carbonate into the lactating cow. Barry (1958) and Sansom and Barry (1958) have reported that most of the lysine, tyrosine, glutamine, glutamic acid, asparagine and proline incorporated into the caseins of goat's milk come directly from the cor- responding free amino acids of the blood. They stated that 70% of essential and 50% of the non-essential amino acids incorporated into casein by synthesis in the goat's mammary gland come directly from the free amino acids of the blood. Peeters g1,al. (1957) injected 835 cysteine into a lactating sheep. They found that the specific activity in the isolated whey proteins was much higher than the specific activity in the casein. The same result was obtained when 835 cysteine was added to the blood during perfusion experiments. This is interesting because it is just Opposite to the results obtained by other workers as reported earlier in this section. A review of the literature on the incorporation of labeled amino acids indicates that almost all of the work reported is of a qualitative nature. It is felt that there is a need for a quantitative investiga- tion on the biosynthesis of milk proteins. The literature on the immune globulins in milk is voluminous and beyond the scope of this review. However, a few of the reports in the literature should be mentioned. Ehrlich (1892) originally found that the colostrum from immune mice transmitted specific antibodies that were absorbed by the nursling. Crowther and Raistrick (1916) reported that there were proteins of a similar nature in both blood and colostrum. Howe (1921) was the first 13 to show that the newborn calf acquires Specific proteins upon ingestion of colostrum. There have been a great many reports confirming these early findings. Smith (1946a, 1946b, 1946c, l9fi6d, 1957) has shown marked similarity between the "r—globulins of the blood and the immune globulins of milk and colostrum by amino acid composition and electrophoretic comparisons. However, he found some differences in amino acid composi- tion. Hansen and Phillips (191+?) and Hansen at al. (19Lp7) have also shown the similarities between these two protein fractions. There are many other reports in the literature supporting the above conclusions. It has been shown many times that the blood of the newborn calf is deficient in 'r—globulins and that after the ingestion of colostrum, 'T-globulins appear very rapidly in the blood of these calves. Recently, Larson (1958) has shown by quantitative electrophoresis that 85 to 107% of the immune globulins of bovine colostrum appear to come from.the blood #92— and ’rl-globulins. Askonas ethal. (l95h) immunized a lactating rabbit and then injected 535 labeled DL-methionine. The antibodies from milk and blood were precipitated by the antigen and counted. The counts were from 7-17 counts per minute.“ The activities in the milk and blood were about the same, but the activities are so low that there is some question about their significance. The previous work certainly indicates a relationship between the blood 7"-globulins and immune globulins in the milk and colostrum. However, the evidence does not preclude the possibility that these ~rLglobulins could have been altered slightly within the mammary gland. Polis etual. (1950) and Coulson and Stevens (1950) have reported "blood" sermm albumin in milk and have indicated that it is very similar to albumin in blood. Larson and Gillespie (1957) have shown by 14 quantitative electrOphoresis that these two proteins are identical. There are no data in the literature on "blood" serum albumin in rabbit“s milk, and it has been investigated in the present study. 5. In_Eitrg Cultivation of Mammary Tissue The cultivation of mammary tissue in_yitzg has been one of the more difficult problems in the area of tissue culture. It has not been until recent years that progress has been made in this area. Hardy (1950) was the first to report the culture of mammary tissue. He reported duct growth but no alveolar develOpment in organ cultures prepared from the ventral body wall of embryonic mice. Lasfargues (1957a, 1957b) has reported proliferation of mammary epithelial cells after the dispersion of the mammary tissue cells by incubation with collagenase. He used various types of serum as a medium. Elias (1957, 1959) and Elias and Rivera (1959) have reported culti- vation of organ cultures from adult mice. They prepared their explants from fully develOped mammary tissue of pregnant mice. The tissues were cultured in a synthetic medium enriched with various hormonal combina- tions at different levels. They found that the addition of estrogen, progesterone, cortisol, growth hormone and prolactin to the media main- tained the mammary explants. Their work was the first successful demonstration of the organ culture of mammary tissue. These workers were also able to initiate some secretion in their explants by means of high levels of cortisol and prolactin. Trowell (1959) has reported the successful organ type cultivation of rat mammary tissue in,yitzg. Larson (1959), Ebner and Larson (1958, 1959), and Hoover 9i a1. (1959) have published abstracts on the cultivation of bovine mammary tissue inpyitzg. .They have been able to grow sheets of mammary I 1 u . I. U‘ ‘1 It»; ‘1' l5 epithelium and maintain this epithelium in culture for many months. Their work is extremely important because of indications of biosynthesis of lactose and fgllactoglobulin by these epithelial cells in culture. Their work indicates the potential of tissue culture techniques for bio- chemical investigations into the synthetic mechanisms of the mammary gland. Tissue culture experiments on mammary tissue are considered to be important because they provide in_yitzg techniques for studies of the biochemistry, endocrinology, and physiology of the mammary gland. This technique also provides a method for the study of the precursors of milk constituents. MATERIALS AND METHODS A. Methods Utilized in the Studies of Rabbit Milk Protein Precursors - 14 l. haze" 0-. 00 o 0‘ '1'... 0-09‘0 - 1.! ' 0 :r o:— - -,oq — -C The radio-chemical purity of Leucine-Z-Clu was ascertained by paper chromatography of a small sample of the material which was purchased from Tracerlab, Inc., 130 High Street, Boston 10, Massachusetts. The amino acid was chromatographed on‘Whatman No. 1 paper by the descending technique. The solvent was 88% liquid phenol:2-propanol:water = 100320320 v/V. The glutamic acid-Z-Clu was purchased from the same company and chromatographed in the same manner. The solvent used was NeButyl alcoholzacetic acidzwater = 25o:6o:25o v/V. The detailed pro- cedures are given by Block and weiss (1956a). The above chromatographs showed a single spot with corresponding Rf values in the proper range. Also, the radioactivity of these strips was located over the individual spots. These determinations were made by means of a gas flow strip counter at the Argonne National Laboratories, Lemont, Illinois. Figure l in the appendix shows a photograph of these chromatographs together with the recording of radioactivity. The specific activities of the leucine-Z-Cli+ and glutamic acid-Z—C14 were 0.51 mc./mm. and 0.54 mc./mm., respectively. Thus, it was estab- lished that the materials to be injected were pure and that the resultant activity in isolated proteins was derived from the injected amino acid. These proteins were produced in,yiyg by the injection of a dose of 11.85 mg. of BaCO3-Clu (1.5 me.) into the marginal ear vein of each of two male New Zeeland white rabbits. The 014 labeled Baco3 was purchased 17 from the Union Carbide Nuclear Company, Oak Ridge, Tennessee. Prior to injection of the labeled BaCOB, 20 mg. of non-labeled BaC03 was injected into rabbits to test the possible toxicity of the barium ion. No effect was noted as evaluated by observation of the smooth muscle for spasms. Immediately following injection of the Cl“ labeled carbonate, the animal was placed in a sealed chamber made out of an ll-gallon aquarium tank. Compressed air was forced through 3 traps and then into the sealed chamber. Starting from the compressed air line, the traps consisted of concentrated H250“, concentrated NaOH, and tap water, respectively. The air was sucked from the opposite end of the chamber by a water aspirator. Three concentrated NaOH traps were placed between the aspirator and the sealed chamber. A flow meter was connected into the system and the air flow was determined to be 500 ml./min. Also, a kerosene manometer was hooked up to the chamber and the input and output of air was regulated to atmospheric pressure. The rabbit was kept in the chamber for 6 hours post injection. During this interval, air samples and samples from the three NaOH traps were taken periodically. There was no 002 buildup and all of the radio- active C02 expired by the rabbit was trapped in the first two NaOH traps. At the end of the six-hour period, the rabbit was removed from the chamber and anaesthetized. A carotid artery and femoral vein were cane nulated. Blood was drained from the carotid artery and physiological saline was run into the femoral vein at a rate of 20 m1./min. This was continued until the animal expired. The majority of the blood clotted shortly after collection in 500 ml. blood bottles. The mixture of clotted blood and blood diluted.with saline was centrifuged and the dilute serum was fractionated by Cohn's at al. (1950) alcohol fraction- ation procedure (Method 10). Because of the dilution of the plasma with l8 saline, slight adjustments of pH had to be made to attain the proper pH's according to Cohn gt_al. (1950). The fractions obtained by the fractionation were albumins, ‘rL globulins, a crude fraction consisting primarily of fg-globulins, and a crude fraction consisting primarily of<9(-globulins. The yields, specific activities, and percent of activity of the injected dose in the protein fractions from.the blood of these two animals are reported in Table l of the appendix. All subsequent blood protein fractions were made in this manner. The 61- andlszglobulins referred to in the re- mainder of this text refer to these crude Cf- andl/Q-globulin fractions. All injected materials were injected via the marginal ear vein. The amino acids and protein fractions were dissolved in a minimal amount of phosphate buffer (pH 7.2). Qxytocin was diluted to a concentration of 1.0 I.U./ml. of distilled water. Blood samples were obtained by heart puncture with a 20 gage needle. An endeavor was made to withdraw the blood from.the left ventricle for the sake of uniformity and consistency of composition. The blood sample was transferred immediately to conical plastic centrifuge tubes and allowed to clot at room temperature. Milk samples were taken from the right and left inguinal glands. Just prior to milking, the animals were injected with one I.U. of oxytocin to stimulate milk ejection from the lobulo-alveolar system. This dose was found to be very effective. The milk was withdrawn in the following way: A vacuum (-15 mm.) was established by means of a water aspirator. A 50 ml. flask was placed 19 in the vacuum line to serve as a milk collecting vessel. A gum rubber tube was attached to the vessel. A beveled glass tube of the proper diameter to easily slip over the rabbit's nipple was inserted into the other end Of the rubber tube. The beveled glass tube was placed on the nipple and the mammary gland vigorously massaged from the periphery toward the base of the nipple. The suction was interrupted every few seconds by removal of the beveled glass tube on the nipple. The pro- cedure was continued until no more milk could be withdrawn from these two glands. The inguinal glands were used because it was found by experience that they were the easiest pair to milk. It is felt that the use of oxytocin materially aided in removal of as much milk as possible from these glands. This was necessary to prevent mixing new milk formed with residual milk and thus causing errors in the determination of the radioactivity of the milk proteins. After milking the animals were returned to their litters. Animals were used only if they had a minimum of four in their litters. This milking procedure was found to be very satisfactory after some experience had been obtained. It was found that extreme nervousness on the part of the rabbit inhibited the release of milk and perhaps milk production also. Therefore, a training period of a few days prior to the beginning of an experiment was helpful. Generally, the first attempt to milk a rabbit was difficult. After a few attempts, the animals be- came accustomed to the procedure. They would usually be very relaxed and no difficulty was experienced. The time required to milk the two inguinal glands as completely as possible was in the order of 15 minutes. At the end of the milking, the 20 blood samples were drawn. During the injection, milking, and bleeding, the animals were restrained on their backs on a small animal surgery board. 4. Ezagtignatign of Rabbit Milk Proteins The rabbit milk was centrifuged at 1°C for 30 minutes to remove the fat. The fat formed a semisolid plug on the surface of the milk. The skim milk was removed by inserting a pipette under the fat and removing the skim.mi1k. This was diluted with an equal volume of distilled water. Caseins were precipitated by adjusting the pH of the skim.milk to pH 4.3 with small additions of 0.5 N HCl. It is of interest that the isoelectric point of bovine caseins is pH 4.7. The precipitated caseins and whey were separated by filtration through Whatman No. 42 filter paper. The immune globulins were at first separated by the method of Smith (1948). During the adjustment of the pH of the whey to 6.0, a gelatinous precipitate appeared in the solution. The precipitation did not increase during the addition of ammonium sulfate to the proper concentration. Paper electrOphoresis of this fraction showed that it had the same mobility as the immune globulin fraction of the original whey when it was subjected to electrophoresis at the same time. Another sample of acid whey was adjusted to pH 6.0. The same precipitate was obtained. No ammonium.sulfate was added. The precipitate was filtered off and subjected to paper electrophoresis. This precipitate from pH adjust- ment only, appeared to be the same as the one to which ammonium sulfate had been added. Therefore, in rabbit milk it appears that the immune globulin fraction can be obtained by pH adjustment only. This protein fraction was homogeneous by the criterion of paper electrophoresis. 21 Therefore, this method was used to isolate the protein fraction referred to as rabbit milk immune globulins in the Results. The c{-lactalbumin fraction was isolated by the method of Gordon and Semmett (1953). /9-1actoglobulin was isolated by the method of Larson and Jenness (1955). The "blood" serum albumin fraction was first noticed on electro- phoresis patterns of rabbit whey. The method of Polis at 31. (1950) was used to obtain the "blood” serum albumin fraction from the rabbit Whey o 5.12:] . EBIJ'IBJi ill']lE!iE H 'When the various fractions were obtained, they were transferred to dialysis sacs prepared from Visking tubing. The fractions were dialized against frequent changes of distilled water until free from.salts, alcohol, and other reagents used in the fractionation procedures. This was usually complete in about 5 days. It was also felt that this pro- cedure might help to remove any free radioactive amino acid which might have adhered to the protein during the fractionation procedures. 6. Laanhi1lizaiian_2£_Eraiein;Ezaaiian§ 'When dialysis was Complete, the fractions were transferred to wide- mouth rat water bottles. The solutions were frozen in a shell of uniform thickness around the inside surface of the bottle. This was accomplished by inserting the bottles into a bath of alcohol and dry ice. As the bottles cooled, they were rotated to facilitate the shelling of the ice on to the surface of the bottle. The bottles were never filled to more than half their capacity. The bottles were attached to the lyophillizer one at a time after the refrigeration bath was cooled to -50°C and high vacuum had been 22 established. A 5-minute interval was used between the placing of samples on the lyOphillizer. This was done to prevent overloading of the appara- tus and loss of vacuum and thus melting of the samples. After the ice had been sublimed and the samples were dried, they were removed and stored for electrophoresis and counting. Paper electrOphoretic patterns were run in Beckman/Spinco paper electrOphoresis cells. The procedure used for developing and staining these blood serum and blood protein fractions is outlined in the manual for the operation of the Beckman/Spinco electrophoresis cell. Rabbit whey and whey protein fractions were first run according to the procedure of Leviton (1957). This procedure was not too satisfactory because the large fl.lactoglobulin fraction with highest mobility in the buffer system used was absorbed on the paper strip along the entire length of the pattern. This absorption masked the peaks of the other whey protein fractions. Due to this difficulty, a method was developed to overcome it. The paper strips were soaked in a 5% glycerol solution containing 0.5% gelatin for 10 minutes. The glycerol was used to prevent the paper strips from.becoming stiff. Next, the absorbed gelatin was fixed on the paper strips by dipping them in 5% formaldehyde for a few minutes. Then the paper strips were washed in tap water and distilled water to remove any excess gelatin and formaldehyde. The strips were dried at 125°C to completely denature the absorbed gelatin. ‘When these gelatin treated strips were used for the paper electro- phoresis of whey proteins, very satisfactory patterns were obtained. This method has been used for all electrOphoresis of whey samples and 23 whey proteins. The gelatin absorbed on the paper strips and denatured by formaldehyde and heat did not stain with bromophenyl blue. It was not detected when the patterns obtained were run through the analytrol. Paper electrOphoretic patterns were run on all serum samples and protein fractions from these samples. - This was also done for whey and whey protein fractions. These paper electrophoretic patterns were utilized to check the identity and homogeneity of the isolated fractions by comparison to patterns run on the original serum or whey. Figure 2 in the appendix is a photograph of a set of paper electrophoresis patterns from an original serum sample, and the fractions isolated from it. Figure 3 in the appendix is a photograph of a set of paper electro- phoresis patterns from whey and the fractions isolated from it. During preliminary investigations, an attempt was made to separate the various protein fractions of rabbit serum and whey by the Continuous Flow Curtain ElectrOphoresis Apparatus. Some separation was achieved but the yields were low. Also, the separation into homogenous fractions was not very good. This was especially true in the case of the blood serum. An equal volume of 20% trichloroacetic acid was added to a small volume of rabbit serum to precipitate the serum proteins. The precipitate was filtered and washed with 3 small portions of distilled water, using Whatman's No. 42 filter paper. The filtrate was extracted 5 times with other to remove the excess trichloroacetic acid. This filtrate was then subjected to column chromatograva by the method of Moore at 5].. (1958) . The filtrate had to be added in small. portions and eluted from the columns a number of times due to the low capacity of the columns. The amount of leucine or glutamic acid in the eluent was detemined colorimetrically. 24 by development of the color in an aliquot with ninnydrin (Moore at al. 1958). The eluent was pipetted into a scintillation bottle and evapo- rated to dryness in a vacuum oven at 70°C. Then it was stored for counting. The amount of free leucine or glutamic acid in the serum was calcu- lated from the following equation: WW=mgongem m1. of original serum sample The mg. % of free leucine in serum samples from animal X-35 (Elcperiment I) is presented in Table 3 in the appendix. The amount of the free blood amino acids in the scintillation bottles was calculated from the following equation: mg. /ml. of eluent x ml. of eluent in the bottle = mg. of free blood amino acid counted It should be remembered that the data reported in the tables in the Results under the heading free blood leucine or glutamic acid counted were determined in the way reported in this section. It is not a weight determined gravimetrically. It is recognized that there are inherent errors in a procedure such as this. However, Moore at al. (1958) report a recovery of 99% on synthetic mixtures of amino acids. It is felt that this procedure is more accurate than attempting to weigh 1 to 2 milli- grams of material. 9. 9‘. :9 ; o e: ‘ u. o. 0' o; 's :0 _;> ,e; :99 - .—III Samples weighing from 40 to 50 mg., when this much material was available, were weighed into small flasks. Twenty milliliters of 20% 501 were added and the samples autoclaved for a minimum of 10 hours at 15 lbs. pressure to hydrolyze the proteins. Ten samples of each blood 25 and milk protein were hydrolyzed in this way. The hydrolysates were neutralized. Aliquots of these hydrolysates were subjected to column chromatography (Moore gt a1. 1958). The leucine and glutamic acid were determined in the eluents by the method of Moore et,al. (1958). The average values for leucine and glutamic acid from these fractions is re- ported in Table 4 in the appendix. 10. W V \3"! 5.01 0 Ant... ,=.9= A; AI' W151 Glutamic acid can be rapidly metabolized to arginine, proline and hydroaqproline. These amino-acids are then available for synthesis into proteins. Also, a major portion of glutamic acid in proteins can be in the form of glutamine. Therefore, it was necessary to isolate the glutamic acid in the protein fractions. If the radioactivity in the whole protein fractions had been counted, it would not have been possible to compare the activities with those found for free blood glutamic acid. Samples of the protein fractions were weighed. A standard amount of 10 mg. of pure glutamic acid was added to each sample. This mixture was hydrolyzed as described in the previous section. The insoluble chloride of glutamic acid was precipitated by cooling the hydrolysate to 0°C according to the procedure of Fischer (1901). The material was dissolved by addition of 0.5 N NaOH and reprecipitated by the addition of 6 N HCl and cooling to 0°C. This material was washed with .01 N HCl and then with acidified alcohol and dried. Paper chromatographs were Pmpared as described earlier and only one spot was evident upon development with ninhydrin. The Rf value was that of glutamic acid. The dried chloride of glutamic acid was weighed into a scintillation bottle for counting. The weight was corrected to the weight of free 26 glutamic acid by the following equation: 1W wt.of free G.A. (mgw. of the chloride of G.A.) The theoretical amount of glutamic acid in the hydrolysate was calculated from the following equation: Theoretical amount of G.A.=(% G.A. in the protein fraction x wt. of the fraction hydrolyzed x lo2 ) + 10 mg. ‘The amount of glutamic acid weighed into the scintillation bottle derived from the hydrolyzed protein fraction was calculated by the following equation: (wt. G.A. in the scintillation bottle) x mg. G‘A. counted = theoretical wt. of G.A. in the hydrolysate The weights of glutamic acid derived from the various protein fractions reported in Tables 7 amd10:ln the Results were determined in this manner. It is obvious that there are many possible sources of error in this procedure. However, it was felt that this procedure would be the most efficient and would provide reliable data. When glutamine is subjected to acids it is converted to free glutamic acid. Therefore, the values reported as free glutamic acid in blood and protein fractions is actually a combination of glutamic acid and glutamine. This is permissible since glutamine is derived from glutamic acid. 11. .2r9cedura_far_ths_Esiimaiion_2f_Eadicaciile_leucins_in_ihe Eno2diand_Milk_E:ciein_Ezaaiicns Leucine is metabolized to acetoacetic acid and acetyl-CoA. The 10.A. 21w. glutamic acid. molecular weight. 27 number 2 carbon of the leucine-Z-Clu becomes the carboxyl carbon in the acetyl portion of acetyl-CoA. Thus, it is evident that this isotopic carbon atom can become incorporated into a large variety of compounds via the Krebs cycle and the pathways for fat synthesis. However, the dilution of this isotopic carbon atom by other metabolic pathways leading to acetyl-CoA is considerable. Also, adequate supplies of amino acids were available from the diet. Because of these considerations, it was not deemed necessary to isolate leucine from the protein fractions. It was felt that the error introduced by radioactivity contributed to the proteins due to the metabolism of leucine and subsequent incorporation into non-essential amino acids would not be any greater than the errors introduced in an isolation procedure for leucine from protein fractions. Forty to fifty milligrams, or as much as was available, of the pro- tein fractions were weighed into scintillation bottles and stored for counting. The weight of leucine in these fractions was calculated from the average percent of leucine in these fractions as determined earlier and reported in Table 4 of the appendix. Thus, the mgs. of leucine counted reported in Tables 1 and 4 in the results is a calculated value based on the percent of leucine in the weighed protein fractions. 14 . 12. .13 'v'U 9-. .9 0 I; SO: , ,‘ fl ' . I. 3i! -_Ct_°9_ W232, These fractions were simply weighed into the scintillation bottles. Samples of 40 to 50 mg. were weighed when this much material.was available. The C1“ would be randomly distributed in the non-essential amino acids of these proteins. Therefore, the activity in these samples was calcu- lated as‘pc./mg. of protein. All samples were counted in the Tricarb )6lliquid Scintillation Spectrometer manufactured by the Packard Instrument Company, Inc., LaGrange, Illinois. This instrument is ideally suited to the counting of carbon-l4 compounds of low specific activity because of its high sensitivity to low energy {flparticles and its very high counting effi- ciency. The samples were weighed into the scintillation bottles as described previously. Two milliliters of hydroxide of hyamine in methyl alcohol were added to the dry proteins or amino acids to take them into solution. The chemical name of hydroxide of hyamine is p—(diisobutyl-cresoxy— ethoxyetnyl) dimethylbenzyleammonium hydroxide. The mixtures were warmed to 50°C in a water bath until the solutions were perfectly clear. Next, 5 ml. of the scintillation fluid was added to the dissolved protein hyamine mixture in the bottles. The scintillation fluid is composed of 6 gm. PPO plus 100 mg. POPOP dissolved in 1 1t. of A.C.S° grade Toluene. PPO is the trade name for (2,5-diphenyloxazole) and POPOP is the trade name for 1,4-diZTZ-(5 phenyloxazole);7 benzene. This scintillation fluid serves the function of transforming the energy of the fl-particle emitted from the unstable one atom into light energy which is, in turn, transformed:hmoeflsctrical energy by the light sensi- tive photocells surrounding the sample bottle. The liquid scintillation system has some disadvantages. The pri- mary problem is that hyamine and other compounds have a quenching effect on the scintillation fluid. This means that some of the disintegrations will not belrecorded because the fluorescence produced is quenched before it can be recorded by the photocells and thus counted by the instrument. 29 The quenching of hyamine was determined by adding a radioactive standard of 2 ml. of hyamine plus 5 ml. of the scintillation fluid. ‘When enough material was available9 samples were prepared in trips licate. Caseins. xg-lactoglobulins, albumins. and in some cases ll globulins and immune globulins were prepared in triplicate. Also, samples of non-radioactive glutamic acid and sodium citrate were dis- solved in hyamine and scintillation fluid added. These samples were spiked with a standard of benzoic acid-C1“ made up in toluene. This standard was prepared to have a theoretical count of th6 disintegra- tions per ml. per minute. Also, bottles were prepared containing hyamine and scintillation fluid only for determining background. The samples were placed in the automatic sample changer in the freezer for a minimum of 4 hours. This time is necessary to cool the samples and to allow for any residual fluorescence due to sunlight to dissipate. The instrument was set up so that each sample was counted for a period of 5 minutes and automatically recorded on a paper tape. The standards plus hyamine counted at an efficiency of 50%. The counts of the duplicate protein samples always counted within 2% of each other. These were averaged and compared to the count of the third. sample plus the spike of 1 ml. of the radioactive standard (10h6 DPM). The spiked sample should have had a count of 523 CPM higher than the average count of its two duplicates since the counting efficiency was 50%. The average difference between the averages of the two protein samples and the spiked sample for all of the samples tested in this way (a total of 50 fractions) was h50.7 : 10.61 higher for the spiked 1The average with standard error. 30 sample. The quenching due to the protein fractions may be calculated in the following way: 523.0 ~ 450.? x 100 = 13.8% 523 This means that 13.8% of disintegrations were not counted due to quench- ' ing of the fluorescence due to the proteins. No quenching was observed in the glutamic acid or citrate samples. The background averaged 11.0 t 1.51 CPMZ. Since the counting efficiency was 50% and the quenching due to pro- teins was 13.8% and background was 11.0 CPM, the following correction factor was applied to convert the raw count of the instrument for the 5-minute period to DPM3 in the case of proteins: DPM : (raw 5 min. count) x 100 x lQO 86.2 x 50 x 5 The amino acids were corrected to DPM by the following equation: _ iraMi5_min._cQunilsx_lQQ DPM - 50 x 5 The term.lQQ§-is dropped from the second equation since no correction is necessary for quenching in the case of the amino acids. The tables in the Results report the activity in terms of pc./mm? for the amino acids. One pc. of an unstable isotOpe is defined as that amount which disintegrates at a rate of 222 x 10“ DPM. Therefore, the pc./mm. are calculated by the following formula: 1The average background with standard error. 2CPM.= counts per minute. 3DPMI= disintegrations per minute. epc./mm. = microcuries per millimole. 31 cho/mmo = WM. 222 x 10 x millimolecular wt. in mg. The millimolecular weights of leucine and glutamic acid in mg. are 131 and 1&7, respectively. The activities of the protein fractions in the tables in the Results are reported as pc./mg. of protein counted. These values may be calcu~ lated in the following manner; DEM pc./mg. of protein counted = u 222 x 10 x mg. of protein counted 1L». W W Table 5 in the appendix presents data on the activity in urine collected on animal X-35 injected with #5 pc. of leucine-Cl“. The animals frequently urinated immediately following the injection of oxytocin. Small samples of the urine were collected when this occurred. The urine was placed in a scintillation bottle, dried and stored for counting. These counts are reported as CPM/ml. of urine. These samples did not yield perfectly clear solutions when treated with hyamine. It is difficult to interpret how much quenching effect this caused. There- fore, the reliability of these data are questionable. Table 2 in the appendix presents a summary of the animals used in the studies of rabbit milk proteins. It describes the animals, materials iJijected into these animals, and amounts of milk and blood withdrawn. 32 B. Methods Used for In 11:29 Studies of Guinea Pig Mammary Tissue LW The proper cleaning of glassware is one of the most important tech- niques in tissue culture. Two problems are encountered: (l) toxicity due to improper rinsing and (2) bacterial contamination due to incomplete cleaning. Micro-801v was used as the cleaning agent for the glassware. This is a special cleaning agent available from Microbiological Associates, Inc. It can be thoroughly rinsed away with water and has very low toxicity properties. It has a pH of 7.0. The glassware is scrubbed vigorously in very hot micro-solv solution until distilled water will run from the glass in a sheet, leaving a thin unbroken film of water on the surface of the glass. Then it is rinsed 5 times with hot tap water, 5 times with regular distilled water, and 15 times with triple glass distilled water. The glassware is then air dried, wrapped in paper towels, and sterilized by dry sterilization at 150°C for one hour or longer. 2. Ezepanatign Q: MEdié Parker's synthetic "199" was used as the basic medium. This medium was originally develOped by Mbrgan §£.filo (1950). This medium contains all the essentials known for the nutrition of cells in,xitzg. It does not contain any hormones. This medium is available in a concentrated form from Microbiological Associates, Inc. The medium contains 10 times the concentration for use and must therefore be diluted. This is accom- plished by placing 41 ml. of triple distilled water in a prescription bottle and autoclaving it for 2 hours at 15 lbs. pressure. After cooling the sterile water, 5 ml. of the stock sterile "199" is removed from its container by means of a sterile needle and syringe and 33 transferred aseptically to the prescription bottle. The pH of this solution is acid. The "199" contains methyl red as an indicator. The pH is adjusted to 7.2 by the aseptic dropwise addition of sterile 1.4% NaHC03. The pH is judged by comparison of the color to methyl red solu- tions of known pH. The medium is then gassed with 95% 02 and 5% 002 by bubbling the gas through the medium by means of sterile glass tube drawn out to a very fine capillary. Sterile stock solutions of hydrocortisone (800 pg./ml.), prolactin (12,000 pg./m1.), insulin (7,000 p-g./ml.), and penicillin (2,500 I.U./ml.) were prepared in advance. One ml. of each of these stock solutions was then added to the medium aseptically. Five milliliters of sterile rabbit serum was then added. This procedure yields 50 ml. of medium II reported in Table 22 of the Results. Medium I was prepared in the same manner, except “2.5 ml. of water is sterilized and .5 ml. of the hydrocortisone, prolactin, and insulin stock solutions are added. Mediums containing radioactive amino acids or protein were prepared in the same manner. However, aseptic technique was not required since the radioactive materials are not sterile. The radioactive materials are added last in the desired concentrations. The medium is then steri- lized by passage through a bacteriological filter and transferred aseptically to a sterile prescription bottle. 3. Iisfine_&nlinzs_usihsd The method of culture used in the tissue culture experiments was the Chen (195“) modification of Fell's (1929) organ culture method, which has been further modified by Shaffer (1956). It was found that the medium tended to evaporate when cultured by this method. This preparation consists of a watch glass supported by moist cotton in a 34 Petri dish. The medium is placed in the watch glass and a treated cellulose acetate raft holding the explants is floated on top of the medium. It was found that the medium evaporated during the culture period. Therefore, the moist cotton was replaced by very wet filter paper pulp. This procedure all but eliminated the media evaporation problem. The completed setups were placed in coffee cans and autoclaved for 2 hours at 15 lbs. pressure to insure sterilization. Just prior to sacrifice of the animal, 1 ml. of the desired media was placed in each watch glass. kWMamamlismmW Non-gravid guinea pigs that had not been suckled for 30 days were used to provide non-secretory mammary tissue for culture. Four to six day postpartum guinea pigs were used to provide secretory mammary tissue for culture. The animals were sacrificed by a blow on the head. The inguinal areas were scrubbed with Roccal solution (800 PPM.) to sterilize the skin. An incision was made in the skin and the mammary tissue removed and placed in sterile basic salt solution. An area of the gland was selected which appeared to have good lobulo-alveolar development. This 'was cut into explants approximately 1 mm. in diameter and thickness. The remainder of the mammary tissue was fixed in Bouin's fluid for h 'hours to serve as a histological control. Three explants were trans- :ferred aseptically to each raft. The rafts were then floated on the Dradia in the watch glass. The cultures were transferred to an incubator (at 37°C. The time required to sacrifice the animals, prepare the ex— Plfiurts and get them into culture was approximately 30 minutes. 0“ .0 A‘n. o "b. A w. iVuu "I u too». 0 “up: n A 35 aw The cultures were checked daily for pH changes in the media. At pH 7.2 the medium.is a deep pink; reddish orange at pH 6.9; and yellow at lower pH's. ‘When the medium changed to a reddish orange color, it was removed by suction into a sterile glass tube drawn out to a fine capillary. Fresh media was added, the raft refloated, and the culture continued. The medium was normally changed every other day. Cultures were normally terminated after 5 days. Cultures terminated prior to 5 days did not show a maximal response to the hormones. Cultures maintained longer than 5 days showed progressively greater degeneration. 6. «bro,- = 00 o - -. 0:0. 9 o 0°4- ' .‘. '00 0 9m. '1. .90. W The explants were transferred to Bouin's fluid for 1 hour for fixa- tion. The larger control tissue was fixed for # hours in Bouin's fluid. The tissues were transferred to 70% alcohol. The control tissue was kept in the 70% alcohol until the explants were ready to be transferred into 70% alcohol. The tissues were dehydrated by running them through the alcohol series and into zylol. They were infiltrated.with low melting point paraffins and subsequently changed to higher melting point paraf- fins. They were finally embedded in 5u-56° melting point paraffin. Care was taken to embed the three explants from the same raft on the same plane in a block. In this way a single section on a slide would contain tissue from all three explants. The blocks were shaped and cooled prior to cutting sections. Sections were cut at 6 p.and fixed on.microsc0pe slides. They were stained with iron hematoxylin and eosin and coverslipped in the usual manner . 36 7. Autoradiographs were prepared by a modification of the method of Gross et,al. (1951). Four slides of each stained tissue cultured in a radioactive medium were prepared in the usual manner but coated with celloidin instead of coverslipping. The slides were coated with a thin layer of Eastman Kodak type NTB emulsion in the dark, placed in a light proof slide box in a horizontal position and stored in the refrigerator. Periodically, these slides were developed to ascertain the extent of ex- posure of the emulsion due to the radioactivity. The time was usually of the order of 2 to 6 weeks. The emulsion was developed for 15 minutes in dektal (Eastman Kodak) at 15°C, then fixed with acid fix for 30 minutes at 15°C. The slides were left in cold running tap water overnight, then dehydrated by running through the alcohol dehydration series. After dehydration, they were coverslipped in the usual way. Longer periods of time for treatment with dektal, acid fix, and washing were found necessary in order to completely'clear the emulsion. This method permits evaluation of the histological condition of the tissue and examination of the autoradiograph on the same preparation. Thus, the concentration of radioactivity can be associated with specific areas of the tissue. The tissue and emulsion are on different planes. Thus, it is possible to examine tissue microscopically, then without changing the field, refocus on the emulsion and study it. The develOp— ment of the emulsion affects the stain by reducing contrast, but not sufficiently so that the tissues cannot be evaluated. 37 8.WWMW§ The alveoli of each explant in 5 fields at high power selected at random were counted. The same procedure was carried out on the control tissues. Thus, a comparison could be made and a very rough estimation of the percent of the lobulo-alveolar system maintained inlxitrg could be calculated. This is reported in Table 24 in the Results. The secretory tissues could not be evaluated by the above manner because of the differences in sizes of the secretory alveoli in the con- trols and explants. Many of the secretory explants contained much larger alveoli than the controls. Therefore, in these explants it was necessary to count the number of non-secretory, secretory, and degenerate alveoli in the 5 fields selected at random. The percent of the lobulo-alveolar system.maintained and the percent of lobulo-alveolar system which were secretory are reported in Table 25. The results are calculated in the following manner: % Of IA; maintained = Iéunainiained x 100 maintained LA + secretory LA + degenerate LA % of LA which was secretory = v§2££2$2£Z_Lé;X 100 maintained LA+secretory LA+degenerate LA It is recognized that this method of evaluation of maintenance and secretion is highly subjective. At best, it is only a very general estimation of the actual condition. It is possible that the average values reported in Tables Zn and 25 in the Results could have an error of 25% or perhaps even more. However, this measure does provide some information on the histological condition of the explants. 1IA: lobulo-alveolar tissue. RESULTS AND DISCUSSION I. Studies on the Precursors of Rabbit Milk Proteins A. EXperiment l. The Incorporation of Leucine-ZmClu into Serum and Milk Proteins by the Lactating Rabbit l. geezease in the Spegifiig Activity of the Extracellnla: Free Leasins_£221 The specific activities of leucine from the various blood and milk protein fractions from X—35 are presented in Table l. The specific actim vity of the free leucine of the blood decreases at an extremely rapid rate during the first 10 minutes post injection. It is evident from an inepec- tion of the data for the specific activities of free leucine in the blood presented in Table 1 and Figure 1 that a model of the biological system operating for the removal of free leucine must be postulated. Rabbit No. X935‘weighed 5.000 gm. If it is assumed that 20% of the body weight is interstitial fluid or extracellular water, then it may be calculated that this extracellular pool is 1.000 ml. If 7% of the body weight is blood and 50% of the blood is plasma, then it may be calculated that the animal had 175 ml. of plasma. The data in Table 3 in the appendix give an average value of 2.6 mg.% of free leucine in the serum. If these values ale assumed to be approximately correct, the following calculations can IDe‘made: 1,000 x .026 = 26 mg. free leucine in HOHexl 175 x .026 = ”.55 mg. free leucine in plasma. A total of 45 no. in 11.5 mg. of leucine was injected into Rabbit No. X-35. “'5 )10. in 11.5 + 26 = 37.5 mg. = 3705/13]. = 0.2863 mo of free leucine in the extracellular pool at 0 time. u5/.2863 = 157.2 pc./mm. in HOHex at 0 time. ¥ 1 HDHex = extracellular water. 39 TABLE 1. SPECIFIC ACTIVITIES OF LEUCINE FROM BLOOD AND MILK PROTEIN FRACTIONS FROM A LACTATING RABBIT (NO. X-3 5, 1“THm17TH DAYS POSTPARTUM) INJECTED WITH “5.0 )JC. OF IiEUCINIB—IZ-mC'L Free Blood Leucine 0.16 12.110 0.19 370.00 2 8,100 0.21 230.00 6 4,660 0.16 170.00 12 2,827 0.17 98.00 24 500 0.18 28.00 36 148 0.21 7.00 48 70 0.20 2.10 72 72 0.25 0.18 Leucine From Casein 2 105,700 4.21 148.00 6 102,200 4.99 122.00 12 51,600 3.65 83.50 24 32 9 “50 3 . 87 “9 . 50 36 13,260 “.80 16.30 48 2,538 4.54 3.30 72 322 5.00 0.38 leucine From Milk 3-Lactoglobulin 2 89,400 2.1 250.00 6 161,700 “.7 200.00 12 979800 3.9 150.00 2“ 27,000 3.2 50.00 36 8,850 “.5 12.00 “8 2,890 5.0 3.“0 72 975 6.3 0.58 Leucine From Milk Ci-Lactalbumin 2 68,900 3.3 120.00 6 81,350 “.0 120.00 12 “2,150 2.7 92.00 2“ 27,300 3.5 “6.00 36 5,110 2.2 1“.00 z+8 3,640 3.8 5.60 1?2 761 “.“ 1.00 lDPM = disintegrations per minute. The calculation of DPM and )1c./mm. eJCplained in the Methods section. 2 q3hle count of this sample was not significantly different from back- ground 0 40 TABLE 1. (CONT.) SPECIFIC ACTIVITIES OF LEUCINE FROM BLOOD AND MILK PROTEIN FRACTIONS FROM A LACTATING RABBIT (NO. X~359 1“TH- 17TH DAYS POSTPARTUM) INJECTED WITH “5.0 pC.OF LEUCINE—Z-Clu hrs. post ini. DPM. mg. leucine counted yc./mm. x.102 Leucine From Milk Immune Globulins 2 11,570 2.“1 28.30 6 31,400 3015 58090 12 26,600 2.96 53.00 2“ 1“,310 2.19 38.60 36 9,130 1.87 28.80 “8 8,610 2.5“ 20.00 72 5,“50 3.28 9.80 Leucine From Blood 'r-Globulins 2 15,650 2.99 30.90 6 25,“90 2.“5 61.“0 12 30.350 3.06 58.50 2“ 13,3“0 1.97 “0.00 36 11,“00 2.28 29.50 “8 13,300 3.10 25.30 72 6,820 3.““ 11.70 Leucine From "Blood" Serum Albumin From Milk 2 705 0.21 20.00 6 1,110 0.26 25.00 12 1,150 0.30 23.00 24 774 0.28 16.00 36 581 0.25 l“.00 “8 “23 0.29 8.60 72 347 0.32 6.40 Leucine From Blood Albumin 2 1“,520 3.90 22.00 6 19,650 “.22 27.50 12 13,220 3.56 21.90 2“' 9.390 3.13 17.70 36 11,800 “.57 1“.90 ’48 6,“55 “.28 8.90 ’72 6,340 4.98 7.50 “1 TABLE 1. (CONT.) SPECIFIC ACTIVITIES 0F LEUCINE FROM BLOOD AND MILK PROTEIN FRACTIONS FROM A LACTATING RABBIT (NO. X-35. 14TH. 17TH DAYS POSTPARTUM) INJECTED'WITH 45.0 pC.0F LEUCINE—Z-Clu leucine From Blood CI-Globulins 2 1.779 1.98 5.31 6 7,880 2.“6 18.90 12 6,“1O 2.80 13.50 2‘IL 2.655 1.59 9.85 36 3,620 2.5“ 8.“l “8 2,818 3.02 5.50 72 2.715 3.47 4.62 leucine From Blood 5— Globulins 2 l,““5 1.15 7.42 6 7,960 1.56 30.10 12 5,680 1.29 26.00 2“ 5,510 1.87 17.“0 36 2,3“0 1.3“ 10.30 “8 2,“95 1.75 8.“0 72 1.892 2.23 5.00 ,4! F- av ' 'H “2 10.0 ,— x Free Blood leucine )‘\ 0 leucine From Casein X 1.0 L- k): 0 O + 0 g 0.1 .. \§i .+ £3 8. 03 \S e +. 0.01 L O + 0.001 , i 1 g g l .L l 0 2 6 12 2“ 36 “8 72 hrs. post inj. FIGURE 1. THE LOG OF THE SPECIFIC ACTIVITY (pc./mm.) VS. TIME (hrs. post inj.) FOR FREE BLOOD LEUCINE AND LEUCINE FROM CASEIN ISOLATED FROM 11.3 5 (LACTATING RABBIT &“TH-17TH DAYS POSTPARTUM INJECTED WITH 45.0‘pc. OF D1..LEUCINE.2.Cl ) :3 “I. :71. I: “3 It may be seen from inspection of the free blood leucine curve presented in Figure 1 that the rate constant (k) for the first 10 minutes (0.16 hrs. post injection) is much greater than the rate constant after 10 minutes. It should be pointed out that the first plot for free blood leucine plot- ted on the graph (Figure 1) represents the specific activity at 10 minutes post injection. The theoretical value at zero time is not represented on this curve. Inspection of the data presented in Table 1 shows that the specific activity of free blood leucine is 3.7 uc./mm. at 10 minutes (0.16 hrs. post injection). The first process from 0 to 10 minutes may be described by the following equations: 1n.—3&z— = k 10 mi . = 1n 0.02 = 1 . " 15702 ( n ) 35 3 73 k = «0.375 min."l t1 = a§93-= 1.85 min. 3 .375 This:means that 37.5% of the leucine was being removed from the extra- cellular pool each minute or that half of the leucine in this pool was exchanged with non-labeled leucine each 1.85 minutes. These data may also be expressed in the following manner: (1) 3.7 x 0.2863 mm. of free leucine in HOHex = 1.06 po. out of the “5.0 no. injected were still in HOHex after 10 minutes. (2) 3.7 x 0.03“? mm. of free leucine in plasma = 0.128“ no. out of the “5.0 no. injected were still in the plasma pool after 10 minutes. Tlduis is based on the assumption that the size of the plasma and HOHex Pools remain constant. 1 0i- new 0 I .‘L . '1 r'v‘ .t. u. ‘ mu ‘I- ._ (I D l; 44 Based on the calculations above, it seems reasonable that during the first few minutes this system is one in which there is a rapid uptake of leucine with little or no return of the labeled leucine into the HOHex pool. It should be pointed out that the extracellular pool is assumed to be composed of the interstitial fluid and plasma. A system such as this can be described by the model for the biological system presented in Figure 2. There are several possible explanations for this system during the first few minutes. Possibilities are that the excess leucine is bound by the reticulo-endothelial system (RES); that some fraction of the plasma protein physically binds this excess leucine; and that this excess is rapidly incorporated into the intracellular leucine pool. It is recognized that the model presented in Figure 2 is a gross oversimplification of the true system. (The assumption is made in the construction of this model that there is an almost instantaneous equilim bration of leucine between the extracellular pool and the plasma pool, and that for practical purposes they may be treated as one pool. The rate constant (k = -0.375 min.‘1), the simple exponential equation, and the t% value (1.85 min.) are undoubtedly made up of at least three individual rates and equations. It appears that this process is so complex that the limited data available are not sufficient to permit a czomplete mathematical treatment of this process. However, the data pre- Sented in Figure 2 might be a reasonably close approximation of the Over-all process. Inspection of the data presented in Table l and Figure 1 reveal trieit from 2 to “8 hours the curve for the decrease in the Specific ac“t:lvity of free blood leucine is almost a straight line. The slight Valrflation in the individual points plotted might be due to experimental HOHex = 21.“5 mg. % mam-$.55'F1105 mg. (4500 PC.) H k = -0.375 min.'1 Plasma HOHin Protein Binding _. -0-375t A - Aoe __J t% = 1.85 min. RES FIGURE 2. MODEL FOR THE REMOVAL OF DL.LEUCINE.2-01” = FROM THE EXTRACELLULAR POOL TO THE LEUCINE POOL OF THE RETICULO-ENDOTHELIAL SYSTEM (RES), THE INTRACELLULAR POOL (HOHin) AND PLASMA PROTEIN BINDING. “5 “6 error. The rate constant for this portion of the curve from 2 to “8 hours and the t% value can be calculated in the following way: 1n 9s9§l.= k(“6 hrs.) = 1n 0.00914 = -4.68 2760 k = —“.68 k = -o.00169 min."l These calculations are interpreted to mean that from 2 hours post in- jection, the net loss in the specific activity of the leucine was 0.169% of the total in the extracellular pool each minute or that the specific activity decreased 50% every “09.5 minutes. It has been stated earlier in this discussion that the extracellular pool of leucine was in the order of 26 mg. prior to the injection of the labeled leucine. If the extremely rapid rate of disappearance of the labeled leucine during the first 10 minutes is considered and also that the lO-minute value for free blood leucine is 2.9 mg.% (Table 3, appendix), then it seems reasonable to assume that the extracellular leucine pool has returned to its normal size by 2 hours post injection. The rabbit in heavy lactation consumes a tremendous quantity of feed. This has varied from 300 to “00 gm. per 2“ hours depending on the individual ani— nml. These animals were fed a standard diet of rabbit pellets which <=ontained a minimum of 18% protein. The average percent leucine in ani- Dual feeds is 2%, Block and weiss (1956b). Assuming that these data are ‘3<>rrect, then it is possible to calculate that the rabbit consumed an a‘Vrerage of l,““0 mg. of leucine per day and absorbed l mg./min. from the Elzft. If it is assumed that the rate constant for the first 10 minutes P0 St injection of the leucine from the extracellular pool remains .“n '9"; ‘- 81.» vi 5 p) . . '“V‘ I. “1 suL v k. v.4 u,‘ x) ix. 5., l ‘ v... {\a '1 .\ “ . 2' ' S V ”<18; “7 l constant (k = -0.375 min.- , t% = 1.85), then this pool is turned over at a rate of 1“.05 mg./min. as determined by the following equation: .3é_.= 4.0 . min. 1.85 1 5 mg / If the same reasoning is applied to this pool after 2 hours, the following value is obtained: 3L: 0 o “09.5 0.063 mg /min Since there is such a drastic change in the decrease in the specific activity of the leucine in this extracellular pool, it must mean that during the latter period of the curve there is a return of the label to this extracellular pool. If the size of the extracellular leucine pool remains constant (26 mg.) and the pool is turned over at a rate of l“.05 mg./min. as calculated from the t% of the first 10 minutes of the curve and also, that the pool is diluted with 1 mg./min. of leucine from the gut, then a return of approximately 13 mg./min. of labeled leucine to this extra- cellular pool must take place. A proposed model for this system is presented in Figure 3. The over-all equation for this process described graphically in Figure 3 is presented along with the model. It is certainly true that the actual equation for this process is highly complex and is composed of at least 3 and in all probabilities more than 3 components. It is so complex and the data so limited that it is impossible to derive the true equation for this process. Careful consideration of these data and mathematics lead toward the idea that perhaps only a small portion of the injected dose (3.7 pc.) behaved in the blood as free leucine and that this was the actual “8 HOHex (26 mg.) Mammary m. —. 1 meme 14.05 W... ——> iii??? tissues,etc. 1 .mi. Bing/An plasma / “I ' k = -0.00169 min.“l Protein / HOHint A = Ace -0.00169 Bindi 1 ng t% = [+0905 mine RES FIGURE 3. MODEL FOR THE TURNOVER OF THE EXTRACELLULAR POOL OF LEUCINE TWO HOURS POST INJECTION. “9 quantity available as a direct precursor of milk protein. The curves after 2 hours appear more like the curves for labeled amino acids administered orally or the curve expected when a colloidal suspension is injected and picked up by the reticulo-endothelial system. If this idea is correct, then a leucine depot must be postulated to account for the data obtained. It appears that a large percentage of this injected leucine was sequestered somewhere in the rabbit's body and was not available for milk protein synthesis. It was then turned over at a rate slightly less than the turnover rate of the extracellular leucine pool. It is postulated that likely sites for this leucine depot might be the reticulo-endothelial system, the intracellular leucine pool, and plasma protein binding. It is reasonable to expect that all of these possibil- ities might be involved. Such a system as this might be expressed in the following way: k1 K k Bound leucine ———4- extracellular-——§-tissue fractions free leucine pool \LRB protein binding The rate of tissue fraction labeling or turnover might simply re- flect the constant (k), plus the biological steady state represented by k1 = k2 + k3 + kn. These rates would be referred to the rate of free leucine in the blood. This discussion has been an attempt to explain and understand the kinetics of the data presented in Table l for the Specific activities of free blood leucine in the rabbit. It was felt that this was necessary and that at least an attempt should be made to determine the parameters 50 for the forthcoming discussion on the precursors of milk proteins. Un- fortunately, this discussion has been seriously hampered by the lack of data on the various pool sizes which make it impossible to completely analyze the curve and make some estimate of the complex exponential equations required to fit the curve presented for free blood leucine in Figure 1. There are many reports in the literature dealing with the adminis- tration of labeled amino acids to animals. Many of these reports describe an extremely rapid uptake of the free amino acid from the blood. Un- fortunately, none of these reports have provided a completely adequate answer to this problem. Henriques at a]... (1955) have reported similar results with glycine in rabbits. They have demonstrated a very high uptake of labeled glycine by the liver. They have subjected their curves to mathematical analysis and postulate at least a three-compartment curve for the early part of their curve as described by Solomon (19“9). The organism does not dis- tinguish between an isotopically labeled metabolite and the natural form of the compound. Since the data in Table l are presented as specific activity, a system or systems for the rapid uptake and replacement of free blood leucine must be postulated for the rapid decrease in specific activity of the free blood leucine. This has been attempted in the above discussion. hThere are a number of possibilities to explain this rapid change of the Specific activity in free blood leucine. These are: very rapid uptake of leucine by the reticulo-endothelial system, mammary tissue, liver, spleen, kidney, lung, intestinal mucosa, interstitial A “‘4' 51 fluid, lymph, plasma proteins, and renal excretion. Friedberg et,gl. (19“8) have reported that 15 minutes after the intravenous injection of S35 labeled methionine into dogs and rats, the activity of the free amino acid in kidney and spleen was higher than the activity of the free amino acid in the plasma. They also reported that the activities in liver and lung were about the same as the activity in plasma and that in 30 minutes the activity of methionine in the proteins of the intestinal mucosa was higher than the activity of free methionine in the plasma. Niklas and Maurer (1952) administered 535 labeled methionine orally to rats and demonstrated a very rapid incorporation into the plasma pro- teins with a corresponding rapid decrease in the specific activity of the free methionine in blood plasma. Tarver and Schmidt (19“2) have also Shown a rapid decrease in the specific activity of free plasma 335 methionine when it was fed to dogs. Abdon and Tarver (1951a, 1951b) in- jected 10 mg. serine-fig-Clu into Long-Evans strain male rats and found a very rapid decrease in the specific activity of free blood serine similar to the results in Table 1. Hughes (195“) has reported that there is a considerable loss of proteins from.the blood due to leakage through the capillaries into the interstitial fluids. This interstitial fluid represents about 20% of the total volume of the mammalian organism or 3 times the volume of blood and thus represents a sizable pool for amino acids. In addition, Hughes states that studies with labeled plasma proteins have shown that these proteins are found in the interstitial fluids and lymph in a very short time and reach equilibrium within a few hours. He further states that the capillary permeability for the free amino acids is 100 times that of the blood proteins. This would bring the amino acids into equilibrium within minutes after the in- jection of an amino acid directly into the circulatory system. Some of 52 the free amino acid would be returned to the general systemic circus lation via the lymphatic system, which may, in part, explain why the rate decrease in specific activity decreases after a few hours. The mammary gland must represent a sizable pool for free amino acids since the specific activity of leucine within caseins, /9-lacto- globulin and C3¥--lactalbumin is quite high. These values at 2 hours post injection are reported in Table l as 1.“8, 2.50 and 1.20 pc./mm. of leucine, respectively. This indicates that there must be a considerable uptake of amino acids by the mammary gland in the rabbit. Hammond and Marshall (1925) reported that the average weight of rabbit mammary glands dehydrated with 95% ethyl alcohol from “0 lactating animals during the first 20 days postpartum was 105 gm. Assuming that the dehydration was 50%, then the fresh weight would be about 200 gm. If 20% of this weight is interstitial fluid, then “0 ml. of interstitial fluid would be present. Graham (1937), Shaw and Petersen (1938), Reineke at al. (1939) and others have shown that there is an arteriouvenous difference of amino acids across the mammary gland. These workers have shown that there is an active accumulation of free blood amino acids by the lactating mammary gland. Therefore, the concentration of amino acids in the interstitial fluid of the mammary gland should be equal to or greater than the con- centration of the corresponding amino acids in the blood. This would represent at least 1 mg. of free leucine. Renal excretion of free leucine could play an important role in the reduction of the Specific activity of free blood leucine, since the level of leucine in the blood at zero time is about 3 times the average value reported in Table 3 in the appendix. The activity of the urine counted at 2 hours post injection is not excessively high, as Shown by 53 Table 5 in the appendix. However9 it is possible that a considerable amount of activity must have been excreted via the urine during the 2-hour period prior to the counting of the first urine sample. This might account for a major portion of the decrease in activity during the first few minutes post injection. It is possible that the concen- tration of leucine in the glomerular filtrate exceeded the renal thresh- old for leucine by the rabbit kidney. Also, there is the possibility that the renal threshold for D-leucine is very low. If this is true, the D form would be selectively excreted and could account for a con- siderable decrease in the specific activity of free blood leucine. It is also possible that the threshold for L-leucine was not reached, since Beyer et a1. (l9h6) have reported that the maximal rate of re- absorption in the kidney of the dog was not reached with loads as high as 26.5 mg./lOO ml. of filtrate. Also, they reported that less than 2% of the filtered amino acid was excreted. If it is assumed that the data for free blood leucine levels pre- sented in Table 3 of the appendix represent an average free blood leucine level, the level of free blood leucine in the blood for the first few minutes post injection is 3 times the normal level. If this is so, the possibility for luxury consumption of leucine into the various pools mentioned previousky exists. This possibility might also help explain the very rapid disappearance of the labeled leucine from the plasma. Judging from the specific activities of leucine incorporated into the milk proteins, caseins, fi?-lactoglobulin and C¥~lactoglobulin reported in Table 1, luxury consumption could not have played too great a role in the mammary leucine pool. That luxury consumption was not too important appears to be true, since the Specific activities of the V n»,- \ ,« ~ "H, tap |,‘ .In a. V1 54 leucine of these proteins decreases at a relatively constant rate, as shown by Figures 1, 4 and 5. If there was an excess of leucine "stored" in some form in the mammary gland available for future synthesis of these proteins, then it would be expected that the specific activity of leucine incorporated into caseins, %?~lactoglobulin and‘9(-lactalbumin would not decrease as rapidly as it does. However, there is some slight suggestion of luxury'consumption, since the specific activity of leucine in these proteins does not decrease as fast as the specific activity of free blood leucine. This is especially evident in the case of CK-lact- albumin (Figure 5) during the first 12 hours post injection. Table 1 presents these specific activities of leucine fromCCYelactalbumin as 1.20, 1.20 and 0.92 pc./mm. of leucine at 2. 6 and 12 hours post injec~ tion, respectively. The above discussion is based on the assumption that the free blood leucine is the direct precursor of the leucine in casein, x9-1actoglobulin and C{-lactalbumin. It appears that luxury consumption is not a very important factor as far as the mammary gland is concerned. However. this does not exclude the possibility that luxury consumption of the injected leucine could be a very important factor in other organs which have been shown to take up large amounts of free blood amino acids, such as the liver, kidney, spleen. lungs, and intestine. Since the decrease in the specific activity of free blood leucine is so great during the first few minutes, it seems reasonable to feel that all of the mechanisms discussed above play an important role in the removal of free leucine from the blood. It is felt that the role of the mammary tissue, reticulo-endothelial system, plasma protein binding, kidney, spleen and liver are of special importance. There are only two possible ways that the specific activity of free blood leucine can be reduced. They are selective removal of the 55 10.?— ) X Free Blood mucine R\§ ::::o . leucine From /9-Lactoglobulin g §. 100" X x g . . 0° .— \. 1 x Q s. ‘8‘ \\ O x 0.01"” o x 0.001 a L I I 1 1 1 O 2. 6 12 2a 36 A8 72 hrs. post inj. FIGURE 4. THE LOG OF THE SPECIFIC ACTIVITY (uC./mm.) VS. TIME (hrs. ost inj.) FOR FREE BLOOD LEUCINE AND LEUCINE FROM -LACTOGLOBULIN ISOLATED FROM x-35 (LACTATING RABBIT 14TH-17TH DAYS POSTPARTUM INJECTED'WITH u5.o ‘pC.OF DL—LEUCINE-Z-Cl )o 56 10.0— x Free Blood leucine "\ ' Leucine From (x-Lactalbumin 1.0- .‘.§ x x E. 001- .S: x Q \ o 3‘ \\ ! 0.01L o v * 0.001 I 1 I L 1 1 I 0 2 6 12 2h 36 48 72 hrs . post inj . FIGURE 5. THE ICC OF THE SPECIFIC ACTIVITY (uc./mm.) VS. TIME (hrs. post inj.) FOR FREE BLOOD LEUCINE AND IEUCINE FROM N-IACTAIBUPEIN ISOLATED FROM x-35 (LACTATINC RABBIT lLLTH-17TH DAYS POSTPARTUM INJECTED WITH 45.0 DC. OF DL-LEUCINE-Z—Cl ,) . 57 radioactive leucine and no removal of the nonelabeled leucine or dilution of the labeled leucine of the blood with nonelabeled leucine. The only possibility for the first pathway is selective excretion of Duleucine by the kidney. At best, this could only account for a 50% decrease in the specific activity if all of the D form remained in the blood and was 100% excreted by the kidney. This ideal situation is not possible in a bio- logical system such as this. The other possibility is absorption of free blood leucine into tissues and organs and absorption of dietary leucine from the gut at an extremely fast rate as discussed previously. This is based on the assumption that the level Of free blood leucine remains relatively constant. Also, the return of nonwlabeled leucine to the general systemic circulation from the lymph during the first minutes would have a dilution effect and should be taken into consideratisn. Humphrey and Sulitzeanu (1958) have shown that the rate of exchange of amino acids between cells and tissue fluids is of the order of 100 mg./ min. and that the size of the intracellular amino acid pool in which this rapid exchange occurs is of the order of 100 times the plasma free amino acid pool. This could most certainly account for a very sizable and rapid dilution of the injected Dlpleucinew2~Clu. Considering all of the above discussion. it is possible to account for this extremely rapid decrease in the specific activity of free blood leucine. However, experiments should be designed to determine what is responsible for this initial rapid decrease in the specific activity of an injected amino acid. The kinetics of this phenomena are not clearly understood at this time. 58 3‘1 111]: id-I :11 . :11'11 Examination of the curves presented in Figures 1. h and 5 for the Specific activities of leucine from caseins, /9-lactoglobulin and Ci-lactalbumin versus time, respectively, show a marked similarity and follow the specific activity of free blood leucine quite well. This is especially true in the case of x9-lactoglobulin. Zilversmit et_al. (1943) have developed the general precursor, product relationships. The theoret- ical curves developed by these workers provide the criteria for decision as to whether the precursor measured is the direct precursor of the product. These criteria are: The specific activity of the precursor must exceed the specific activity of the product until the specific activity of the product reaches its maximum, at which time the two speci- fic activities become equal and thereafter the specific activity of the product is greater than the Specific activity of the precursor. The theoretical curves for precursor and product as outlined by Zilversmit .fii.filo (1943) are different from the curves presented in Figures 1, h and 5. This is to be expected because the product is constantly being removed from the system and there is no turnover rate for these products. These three curves fit the criteria for precursor and product quite well. The curve for l9-lactoglobulin matches the criteria almost perfectly. 'When the area under the blood curve is compared with the area under the milk protein curve, it becomes evident that the areas are quite similar. If only one component in the blood is significantly labeled and it is acting as a direct precursor of the milk protein, then the area under the specific activity curve of free blood leucine should equal the area under the Specific activity curve of leucine from the milk proteins. Dividing the area under the free leucine of blood curve into the area .o. .1- R’V In 1“. l4 5" 'u 51 ‘1 o- t V 59 under the milk curve should give the percent of leucine in the milk pro- tein derived from the free leucine of blood plasma. In these 3 cases, it is evident that this would give a value greater than 100%. This is attributed to a time lag. From inspection of these curves it becomes evident that the Specific activity of leucine in the milk protein iS equal to the specific activity of the free blood leucine 2 to 6 hours earlier. An example may help to clarify this theory. The Specific activity of the leucine of /9-lactoglobulin (Figure 4) at 32 hours post injection is 0.2 pc./mm.; at 28 hours post injection, the Specific activ- ity of free blood leucine is also 0.2 pc./mm. This can be interpreted to.mean that it takes an average of 4 hours for leucine to move from the plasma pool to the free leucine pool in the mammary gland and then synthesized into one of these milk proteins and then into the lumen or duct, ready to be excreted as a milk constituent. Also, the high values could be due, in part, to residual milk left in the lumen of the alveoli after milking, which would tend to cause higher than actual values at the next milking. This might also explain the irregularities observed in some of the curves presented. Barry (1952) has carried out similar experiments on lactating goats. He has injected goats with (P32) Na3P04 and determined the specific activity in blood and casein at various time intervals. The work of Aten and Hevesy (1938) and Colas at al. (1950) Showed conclusively that all of the phOSphoruS in casein is derived from free phosphate of the blood. Barry reasoned that the areas under these two curves for blood phosphorus and casein bound phosphorus should be equal. ‘When he divided the areas, he found that the experimental value was 70% instead of the theoretical 100%. Therefore, he has reported that this treatment of the data will give a value within.f30% of the true value. If Barry's work 60 is correct, then it can be concluded that most of the leucine in caseins, /§Llactoglobulin and C’C-lactalbumin synthesized by this rabbit was derived directly from the free leucine in the blood; also, that the leucine in tha mammary pool is derived from the leucine in the blood and is not con— tributed to by other sources to any major extent. Barry (1952, 1956), Sheldon-Peters and Barry (1956), Sansom and Barry (1958), and Barry (1958) have obtained evidence by similar experi- ments on goats that lysine, tyrosine, glutamine, glutamic acid, asparagine and proline of goats' casein is derived to a large extent from the corres- ponding free amino acid or amide of the blood. They have also shown that at least 70% of the essential amino acids and at least 50% of the non— essential amino acids are derived from the corresponding free amino acids of the blood. They have further shown that a very small proportion of the non essential amino acids are derived from glucose within the mammary tissue. Campbell and Work (1952), Askonas et a1. (1954), and Askonas gt,al. (1955) have done similar experiments on the rabbit and goat. These workers have shown that the valine, lysine, glycine, and methionine of casein and /9-lactoglobulin are derived directly from the blood. They have also shown that the plasma protein activity is approximately 10% of the activity present in casein and [gtlactoglobulin at‘6 hours post in- jection in the rabbit and goat. They have also been able to demonstrate that the labeling in these proteins was uniform by partial hydrolysis, isolation of the various peptides and counting these peptides. Larson and Gillespie (1957) have injected (01“) sodium carbonate into a cow and measured specific activities in the milk proteins. They found high specific activities in C(- and x9-casein, /5Llactoglobulin and <={-lactalbumin which were about equal. This suggests, also, that these proteins were formed from free amino acids in the blood. Kleiber at al. 61 (1952) have also reported similar results. It has been shown by this work that the leucine incorporated into rabbit milk caseins, A9-1actou globulin and C(-lactalbumin was derived from leucine that existed some hours earlier as free blood leucine. These results are in very good agreement with the results of others on other amino acids and other lactating mammals. h. T at t e n -G l'n of th B 0 th G \b n 0 Mi Inspection of the curves for the specific activity of leucine from 'Ylglobulins of the blood and immune globulins of the milk presented in Figures 6, 7 and 8 indicate a remarkable similarity between them. They are almost identical and there is no significant difference between them. It is quite obvious that the curve for immune globulins from milk differs to quite an extent from the curves presented for caseins, CX-lactalbumin and Ag-lactoglobulin. This indicates that free blood leucine could not have been a direct precursor of leucine in immune globulins from milk since it has been pointed out in the previous section that leucine of caseins, CXrlactalbumin and AT-lactoglobulin are derived from this source. These data on blood. ?Lglobulins and immune globulins indicate that the relationship between them is not that of a precursor and product since they do not meet the criteria of Zilversmit at al. (19b3). It appears that these two protein fractions are extremely similar when judged on 'the basis of the specific activity of leucine incorporated into them by the lactating rabbit. Table 2 and Figure 9 present the ratios of the specific activities °f7 leucine from milk immune globulins to the specific activities of blood 71- globulins from samples taken from the animal at the indicated times 62 1.000P X Free Blobd leucine X\ o leucine From Blood x holobulins ? " \° S .\ . <3 0.1' i x E“ \ x 0.01- a: 0.001 1 1 l l a 1 l 0 2 6 12 2h 35 1+8 72 hrSo p081: 1.an FIGURE 6. THE LOG OF THE SPECIFIC ACTIVITY (um/mm.) vs. TIME (hrs. post inj.) FOR FREE BLOOD LEUCINE AND LEUCINE FROM BLOOD Y-GLOBULINS ISOLATED FROM x-35 (LACTATINC RABBIT 14TH-17TH DAYS POSTPARTUM INJECTED mm 15.0 pC. OF DL-LEUCINE-Z-Cll'i. 63 10.0- N X Free Blood leucine *\\\\ O Leucine From Milk Immune & Globulins 1,0... \x .\. O x\. \. E \L\ \. 0.1!. C Q i x 01 \o O x 0.01- v ,. 0.001- ll III I l I l 1 o 2 6 12 21+ 36 48 72 hrs. post inj. FIGURE 7. THE LOG OF THE SPECIFIC ACTIVITY (pm/mm.) vs. TIME (hrs. post inj.) FOR FREE BLOOD LEUCINE AND LEUCINE FROM MILK IMMUNE CLOBULINS ISOLATED FROM x-35 (LACTATINC RABBIT lam—Hm DAYS POSTPARTUM INJECTED WITH 1+5.0 pC. OF DLLEUCINE.2-C1 ). 6h 10.0 p X leucine From Blood ‘rLGlobulins ' Leucine From Milk Immune Globulins 1.0—- :O\ X\ i X ' ~===:::::::::X ‘ O E \ x g 0915- . 63 .52 0.01 . I 1 L l I 1 L 2 6 12 2h 36 #8 72 hrs. post inj. 0.001 0‘ FIGURE 8. THE LOG OF THE SPECIFIC ACTIVITY (pm/mm.) VS. TIME (hrs. post inj.) FOR LEUCINE FROM BLOOD hGLOBULINS AND LEUCINE FROM MILK IMMUNE GLOBULINS ISOLATED FROM X-35 (LACTATING RABBIT Infill—17TH DAYS POSTPARTUM INJECTED WITH 1+5.O pC. OF DL-LEUCINE—Z-C . TABLE 2. 65 RATIOS OF THE (a) SPECIFIC ACTIVITIES 0F LEUCINE FROM MILK IMMUNE GLOBULINS TO THE (b) SPECIFIC ACTIVITIES OF LEUCINE FROM BLOOD 7O-GLOBULINS FROM LACTATING RABBIT (N0. x-35, lHTH-l7TH DAKS POSTPARTUM) INJECTED WITH u5.0 p0. 0F LEUCINE-2-C? . hrs. % of milk immune globulins post inj. a/b derived from blood ‘YLglobulins 2 0.916 91.6 6 0.959 95.9 12 0.906 90.6 2n 0.966 96.6 36 0.977 97.7 #8 0.791 79.1 . 72 0.837 83.7 0.909 f 0.1%1 l The mean of the ratios is presented with standard error. 66 1.00_.—-——— —— ————-——— —_ -- -_ -_ __ _- __ _ __ /.\ O . O Q \.-__’~______. 0.75” 0.50— 0.25— 0.00 1 l I I 1 I J 0 2 6 12 214 36 1+8 72 hrs. post inj. FIGURE 9. RATIOS OF THE SPECIFIC ACTIVITIES OF LEUCINE FROM MILK IMMUNE GLOBULINS TO THE SPECIFIC ACTIVITIES OF LEUCINE FROM BLOOD 7c. GLOBULINS ISOLATED FROM x-35 (LACTATING RABBIT lLITH_17TH DfiYS POSTPARTUM INJECTED WITH 15.0 pC. OF DL-IEUCINE—Z-Cl ) 67 post injection of 1eucine~2~C14. These data indicate that the system was probably in equilibrium at 2 hours post injection, the time when the first milk sample was taken. The average of these ratios is 0.909 f 0.174. This indicates that during the period of this experiment, at least 73.5% of the immune globulins secreted into the milk by this animal came directly from blood 7’Lglobu1in. The data presented in Table 2 indicate an efficient mechanism for the tranSport of this pro- tein fraction from the vascular system to the interstitial fluid and then into the epithelial cells of the mammary gland and thus into the milk. This is indicated because the system is already in equilibrium sometime prior to 2 hours post injection. An efficient mechanism for transport of ‘Ylglobulin is also indicated by the relatively high molecular weight of rabbit blood 'rLglobulin of 160,000, which has been determined by Nichol and Deutsch (1998). The data indicate very strongly that the immune globulins were not synthesized within the mammary gland. This is supported by the findings of Humphrey and Sulitzeanu (1958), who injected (Clu) amino acids into rabbits to locate the sites of synthesis of antibodies. They found labeled antibodies in lung, bone marrow, spleen, and lymph nodes. They did not find labeled antibodies in liver, kidney, or'mammary tissue. Askonas and Humphrey (1958) have shown that cells from various tissues are capable of synthesizing antibodies and other 'YLglobulins in,yitrg. The spleen, lung, and bone marrow were shown to be the most important sites of synthesis. There is voluminous literature on the immune globulins of bovine milk. Ehrlich (1892) was the first to detect antibodies in colostrum. Smith (19H6a, 1946b, 1946c, 1946d, 19“?) has done a tremendous amount of work on the physical characteristics and chemical composition of 68 milk immune globulins and blood hglobulins. He has been able to demonstrate quite a close relationship between the two protein fractions but has shown some differences in amino acid composition. This is par- ticularly true in the case of leucine. Smith (1948) reported in his review on immune globulins in cowgs milk that his average values for the percent of leucine in euglobulins, pseudoglobulins and ~V-globulins were 10.4, 9.1 and 7.4, respectively. This is approximately a 20% difference between immune globulins of cow's milk and 'Ylglobulins. This would mean that a considerable amount of leucine is incorporated into the 73-globu1ins of blood by the mammary gland prior to their secretion in the milk. The data in Table 1 do not substantiate this process in the case of the lactating rabbit. However, it is probable that the specific activity of free leucine in the mammary gland and the specific activity of free leucine being utilized for synthesis of 1’4globulins at other sites in the body are very similar. If this is true, then determinations of specific activity of leucine will not provide information on the incorporation of additional leucine into ‘71globu1ins by the mammary gland. There are inherent difficulties in amino acid determinations on protein hydrolysates which make it difficult to state positively that these proteins were identical. Hansen §1_al. (1947) have also shown a close similarity between the amino acid composition of_a purified pseudo- globulin and human ‘Y;globulin. Studies by Jameson at a]... (1942), San Clemente and Huddleson (1943) and by Hansen and Phillips (1947) have shown conclusively by electro- phoresis that the blood of the newborn calf is deficient in the ‘YLglobulin fraction. Hansen and Phillips (1947) and Hansen et_al. (1947) have shown that this ‘Ylglobulin appears very rapidly in the blood of the newborn 69 calf if colostrum is fed during the first 24 hours postpartum. These electrOphoretic studies demonstrated very similar mobilities for the blood 'Ylglobulins and the immune globulins of milk. Mbre recent studies by Larson and Kendall (1957) by quantitative electrOphoresis have shown that the A32- and 'Yjeglobulins build up in the blood stream several weeks prior to parturition and then drOp significantly as the colostrum is being formed. Larson (1958) has shown by quantitative electrOphoresis that the immune globulins found in the mammary secretions can account for 85 to 107% of the ,8 _ and rl-globulins lost from the blood. 2 Studies with isotopes by Campbell and werk (1952), Kleiber 23.810 (1952), Black and Kleiber (1951+). Askonas at a1. (1954), and Larson and Gillespie (1957) have indicated that there are two types of labeling which occur in milk proteins in the lactating bovine, goat and rabbit: high specific activities which indicate that these proteins are derived from free amino acids of the mammary pool which is in equilibrium with the blood free amino acid pool, and the other type of labeling is a low specific activity in the order of magnitude of one-tenth that of the high activity. The data of these workers suggested that the low activity fraction consists primarily of immune globulins and that it comes directly from the blood without alteration in the mammary gland. Askonas etflgl. (1954) immunized a rabbit with formalin—treated pneumococcus type III. The animal was then injected with 25 pc. of S35 labeled DL—methionine. He isolated the antibodies from milk and blood samples by precipitation with the antigen. He found a very close correlation between the activ- ities in the antibodies from milk and the antibodies from blood. However, the activities of their samples were very low. They were from 7 to 17 counts/min./0.3 sq. cm. of infinite thickness. These activities are so 70 low that it is difficult to evaluate the significance of these data and to state positively that they were derived from the same source. The data in Table 1 show that the counts of the milk immune globulins and blood 'Ylglobulins are in the order of thousands of counts per minute and are therefore much more significant. Many workers have demonstrated the striking similarity between blood hglobulins and milk immune globulins in the bovine. Larson (1948) in particular has demonstrated the quantitative relationship between the two in the bovine. The data presented in Table 1 and Figures 6, 7, 8 and 9 show quantitatively that at least 73.5% of the immune globulins of rabbit milk are derived directly from the blood Y-globulins. These data further show that the milk immune globulins were not synthesized within the mammary cells because of the dissimilarity between the specific activities of leucine from milk immune globulins and the specific activ- ities of leucine from casein, #9-lactoglobulin and CX-lactalbumin which have been shown to come directly from the free amino acid pool of the mammary gland which is in equilibrium with the free amino acid pool of the plasma. Furthermore, the results on the rabbits are in agreement with the results obtained on the bovine. They also substantiate the work of Askonas at al. (1954) on the rabbit and are believed to be more reliable due to the much higher level of activity. Also, it has been possible to quantitate these data and they agree quite we11.with the results obtained by Larson (l958)on the cow. 5. I} 9.: E"EJ 3"5 El] . i BH'!IIH] Table 1 presents the data on the specific activities of leucine from "blood" serum albumin from milk and blood albumin. Again, there is a very close resemblance between the labeling in these two proteins. 71 Figures 10, 11 and 12 demonstrate graphically the extremely close similarity between them. This case appears to be the same as the situ- ation between blood ‘Vlglobulin and immune globulins from milk. On the basis of these data, it appears that the blood albumin and "blood" serum albumin of milk are identical since their activities are so similar. Table 3 and Figure 13 present the ratios of the specific activities of leucine from "blood" serum albumin from milk to the specific activities of leucine from blood albumin. They Show that the ratios are quite close to one. The mean of the ratios indicates that at least 77.4% of the "blood" serum albumin in milk from this rabbit comes directly from the blood albumin during this 72 hour period. The equilibrium was reached very rapidly. From the data, it appears that it was reached in less than 2 hours. These results are in very close agreement with the results for immune globulins from milk and blood )7eglobulin. This "blood" albumin from rabbit milk has not been reported previously in the literature. These are the first data of either a qualitative or quantitative nature on "blood" albumin from rabbit milk. Larson and Gillespie (1957) have reported this protein in bovine milk to be the same as blood albumin. Earlier, Polis at al. (1950) reported a method for the separation of "blood" serum albumin from cow's milk and reported that its physical characteristics were the same as blood albumin. Also, Coulson and Stevens (1950) have shown that milk and blood albumin of the cow are serologically identical. These data on "blood" serum albumin from rabbit milk are in good agreement with the data on the cow. 6. S f c A.t + c to 5a__aEd_42_filehulina_efl_fllaei Table l and Figures 14 and 15 also present the Specific activity in the x 0.001“ I L I I I I j 0 2 6 12 24 36 48 72 hrs. post inj. FIGURE 24. THE 100 OF THE SPECIFIC ACTIVITY (pm/mm.) VS. TIME (hrs. post inj.); FOR FREE BLOOD LEUCINE AND LEUCINE FROM "BLOOD" SERUM ALBUMIN FROM MILK ISOLATED FROM L32 (LACTATING RABBIT 14%; 17TH DAYS POSTPARTUM INJECTED WITH 45.0 p0. DL-LEUCINE-Z-C ). I\\.\\\\,\\\I.I~\.V\ \i.knb\ 9.5 10.0 F X Leucine From Blood Albumin 0 Leurine From "Blood" Serum Albumin From Milk 1.0" + E /O;‘\tx .\ E I \X\ \ I o\x U .\$\x l 0.1 U. \K. 03 Q \\ 0.01 ‘ l l J l l I 0.001 I 2 6 12 24 36 48 72 o-—-—=; hrs. post inj. FIGURE 25.. THE LOG 0F SPECIFIC ACTIVITY (pc./mm.) VS. TIME (hrs. post inj.) FOR LEUCINE FROM BLOOD ALBUMIN AND "BLOOD" SERUM ALBUMIN FROM MILK ISOLATED FROM L32 (LACTATING RABBIT 14TH-1ZTH DAYS POST.-. PARTUM INJECTED WITH 45.0 110. OF DL..L.EUCINE.2..Cl ). 96 F- O 1.00 —’\~ -——.—----- -——————— — —-—— _ _ ._____ \. ./ \._fi 4o——4f—o 0.75 *" 0°50 '— 0.25 I- 0.00 I I I I I l I 0 2 6 12 24 36 48 72 hrs. post inj. FIGURE 26. RATIOS OF THE SPECIFIC ACTIVITIES OF IEUCINE FROM "BLOOD" SERUM ALBUMIN FROM MILK TO THE SPECIFIC ACTIVITIES OF IEUCINE FROM BLOOD ALBUMIN ISOLATED FROM X—32 (LACTATING RABBIT 14TH-1ZTH DAYS POSTPARTUM INJECTED WITH 45.0 PC. OF DL-LEUCINE-Z-Cl ). Ilvk..\ \.\\\ 5.: AI! \ \ 97 10.0 '- X Free Blood leucine 1r \ 0 Leucine From Blood “' o(«Globulins 1.0 '- X S‘ -\. € \0 L3 3. 0.1 - . m \ \0 x .\ x \. i 0001 r X\ 0.001v I I I + l 1 1' 0 2 6 12 24 36 48 72 hrs. post inj. FIGURE 27. THE LOG 0F SPECIFIC ACTIVITY (pm/mm.) VS. TIME (hrs. post inj.) FOR FREE BLOOD LEUCINE AND LEUCINE FROM BLOOD d—GLOBULINS ISOLATED FROM X—32 (LACTATING RABBIT 14TH-17TH DAYS POSTPARTUM INJECTED WITH 45.0 no. DL.L.EUCINE_2.Cl ). 98 1000 - Ar x Free Blood Leucine \x ' Leucine From Blood 18 -Globulins 1.0" /03 lac/mm O 7.4 I 0.01 r 5‘ p 0.001 4 I I I I 0 2 6 12 24 36 48 72 hrs. post inj. FIGURE 28. THE L0G OF THE SPECIFIC ACTIVITY (mu/mm.) vs. TIME (hrs. post inj.) FOR FREE BLOOD LEUCINE AND LEUCINE FROM fi-GLOBULINS ISOLATED FROM L32 (LACTATING RABBIT 14TH..17TH DAYS POSTPARTUM INJECTED WITH 45.0 p0. 0F DL-LEUCINE-Z-Cl ). 99 TABLE 7. SPECIFIC ACTIVITIES OF GLUTAMIC ACID FROM BLOOD AND MILK PROTEIN FRACTIONS FROM A LACTATING RABBIT (NO. X-30, 14TH~17TH1BAYS POSTPARTUM) INJECTED WITH 150 DC. DE GLUTAMIC ACID-Z-C hrs. post inj. DPM mg. glutamic acid counted pc./mm. x 10 Free Blood Glutamic Acid 0.16 35.700 0.100 250.00 2 8,137 0.070 76.80 6 4,019 0.050 53.20 12 1,411 0.047 19.80 24 528 0.049 7.10 36 252 0.050 3.30 48 102 0.044 1.50 72 61 0.100 0.40 Glutamic Acid From Casein 2 152.150 1.100 91.60 6 126,900 1.000 84.00 12 660.500 11.100 39.10 24 257.000 10.400 16.40 36 152.200 10.000 10.80 48 48.390 9.700 3.29 72 27.695 10.100 1.81 Glutamic Acid From Milk fl-Lactoglobulin 2 158,500 1.00 105.00 6 122.500 1.03 78.90 12 96,500 1.10 58.10 24 308,000 8.50 24.00 36 127,000 9.70 8.60 48 69,000 10.00 4.56 72 18,300 6.60 1.21 Glutamic Acid From Milk c(-Laota1bumin 2 645,000 7.80 54.75 6 585,000 8.40 46.10 12 453,000 8.10 37.00 24 246,000 7.90 23.10 36 102,500 7.80 8.72 48 36,900 7.70 3.18 72 13,470 7.80 1.14 100 TABLE 70 (CONT.) SPECIFIC ACTIVITIES OF GLUTAMIC ACID FROM BLOOD AND MILK PROTEIN FRACTIONS FROM A LACTATING RABBIT (NO. X—30, 14TH~17T§4DAYS POSTPARTUM) INJECTED WITH 150 EC. OF GLUTAMIC ACID-2_C hrs. post inj. DPM mg. glutamic acid counted pc./mm. x 10 Glutamic Acid From Milk Immune Globulins 2 9,140 5.40 1.121 6 22,485 4.90 3.04 12 18,800 4.70 2.65 24 14,250 5.00 1.89 36 8,610 4.10 1.39 48 6,715 4.40 1.01 72 39380 2.90 0.77 Glutamic Acid From Blood 'YLGlobulins 2 9,165 4.70 1.29 6 12,400 2.60 2.92 12 8,480 2.00 2.80 24 6,780 2.30 1.95 36 7,815 3.50 1.48 48 6,495 3.90 1.10 72 7,850 6.20 0.84 Glutamic Acid From Blood Albumin 2 23,900 8.30 1.89 6 369900 7090 3009 12 25,800 6.80 2.51 24 22,400 8.50 1.74 36 12,600 8.00 1.04 48 9,960 7.20 0.92 72 5,200 6.90 0.50 Glutamic Acid From "Blood" Serum Albumin Fraction From Milk Serum 2 63 0.030 1.39 6 107 0.270 2.62 12 71 0.020 2.34 24 66 0.025 1.74 36 342 0.023 0.99 48 20 0.020 0.66 72 45 0.060 0.50 1 This value is not significantly different from the specific activity of blood ‘Y4globulins at 6 hours. 2The count of this sample was not significantly different from background. TABLE 7. (CONT.) 14TH-17T ACID-2-C 101 SPECIFIC ACTIVITIES OF GLUTAMIC ACID FROM BLOOD AND MILK PROTEIN FRACTIONS FROM A LACTATING RABBIT (NO. X-30, §4DAYS POSTPARTUM) INJECTED WITH 150 p0. OF GLUTAMIC hrs. post inj. DPM mg. glutamic acid counted chmm. x 10 Glutamic Acid From Blood CK-Globulins 2 12,500 4.70 1.76 6 359390 8.30 2.72 12 17.590 3.70 3.15 24 32,190 9.60 2.10 36 14,100 5.00 1.87 48 5,290 4.50 0.80 72 6,690 7.70 0.57 Glutamic Acid From Blood f9-Globulins 2 24,400 4.70 3.44 6 23,000 2.60 5.85 12 17,830 4.10 2.88 24 10,710 4.20 1.69 36 8,740 4.50 1.27 48 5,110 3.30 1.02 72 4,670 6.20 0.50 TABLE 8. 102 RATIOS OF THE (a) SPECIFIC ACTIVITIES 0F GLUTAMIC ACID FROM MILK IMMUNE GLOBULINS TO THE (b) SPECIFIC ACTIVITIES 0F GLUTAMIC ACID FROM BLOOD 1’;GLOBULINS FROM A LACTATING RABBIT (N0. x-30, 14TH-17TH DAYS POSTPARTUM) INJECTED WITH 150 p0. OF GLUTAMIC ACID-Z-C hrs. % of immune globulins derived post inj. a/b from blood 1';globulins 2 0.868 86.8 6 1.040 100.0 12 0.948 94.8 24 0.970 97.0 36 0.939 93.9 48 0.918 A 91.8 72 0.922 92.2 1 0.943 f 0.0141 1 The mean of the ratios is presented with standard error. 103 TABLE 9. RATIOS OF THE (a) SPECIFIC ACTIVITIES OF "BLOOD" SERUM ALBUMIN FROM MILK SERUM TO THE (b) SPECIFIC ACTIVITIES OF BLOOD ALBUMIN FROM A LACTATING RABBIT (NO. X-30, 14TH—17TH DAYS POSEPARTUM) INJECTED WITH 150 pC. OF GLUTAMIC ACID—2.01 hrs. % of "blood" serum albumin from milk post inj. a/b serum derived from blood serum albumin 2 0.735 73.5 6 0.850 85.0 12 0.933 93.3 24 0.998 99.8 36 0.948 94.8 48 0.722 72.2 72 1.000 100.0 0.884 1 0.03861 The mean of the ratios is presented With standard error. 104 TABLE 10. SPECIFIC ACTIVITIES OF GLUTAMIC ACID FROM BLOOD AND MILK PROTEIN FRACTIONS FROM A LACTATING RABBIT (NO. X-34, 14TH- 17TH DAYS POSTPARTUM) INJECTED WITH 150 pC. OF GLUTAMIC ACID.2-C1“ hrs. post inj. DPM mg. free glutamic acid counted pc./mg. x 10 Free Blood Glutamic Acid 0.16 35.200 0.110 209.000 2 14,050 0.090 103.400 6 5,560 0.078 47.300 12 1,950 0.049 26.350 24 648 0.040 10.700 36 501 0.055 3.320 48 131 0.046 1.890 72 159 0.130 0.809 Glutamic Acid From Casein 2 270,500 1.500 119.000 6 131,300 1.300 66.800 12 606,500 10.500 38.200 24 231,500 9.800 15.600 36 87,550 8.600 6.730 48 56,900 10.200 3.690 72 19,490 10.900 1.180 Glutamic Acid From Milk f9-lactoglobulin 2 151,100 1.100 91.000 6 248,000 2.500 65.700 12 373.000 5.300 46.600 24 392,000 9.800 26.500 36 151,100 10.500 9.950 48 62,650 7.600 5.550 72 24,800 4.900 3.350 Glutamic Acid From Milk 0(-Lactalbumin 2 668,000 8.900 49.700 6 391,900 5.700 45.500 12 396,000 7.300 35.900 24 165,800 6.800 16.100 36 85,900 9.100 6.250 48 38,350 6.500 3.910 72 19,550 8.400 1.540 105 TABLE 10° (CONT.) SPECIFIC ACTIVITIES 0F GLUTAMIC ACID FROM BLOOD AND MILK PROTEIN FRACTIONS FROM A LACTATING RABBIT (N0. x.34, 14TH-17TEnDAYS POSTPARTUM) INJECTED WITH 150 pC. 0F GLUTAMIC C ACID—2- hrs. post inj. DPM mg. free glutamic acid counted uc./mg. x 10 Glutamic Acid From Milk Immune Globulins 2 13,100 6.100 1.425 6 20,300 4.700 2.860 12 20,600 5.000 2.730 24 10,180 3.300 2.010 36 7,400 2.900 1.690 48 7,870 4.200 1.240 72 5,765 5.800 0.659 Glutamic Acid From Blood IC-Globulins 2 11.990 5.100 1.550 6 16,000 3.500 3.020 12 12,700 2.900 2.900 24 13,680 4.000 2.260 36 7,230 2.600 1.840 48 6,560 3.300 1.317 72 7.840 6.700 0.775 Glutamic Acid From Blood Albumin 2 20,690 8.000 1.710 6 24,850 5.900 2.790 12 21,400 6.300 2.250 24 16.410 6.800 1.580 36 11,200 7.500 0.990 48 10,780 8.600 0.830 72 7,210 8.100 0.590 Glutamic Acid From "Blood" Albumin From Milk Serum 2 62.7 0.029 1.430 6 122.1 0.031 2.610 12 63.2 0.020 2.090 24 37.9 0.017 1.480 36 35.91 0.025 0.950 48 21.5 0.018 0.790 72 46.1 0.050 0.610 1 The count of this sample was not Significantly different from background. 106 TABLE 10. (CONT.) SPECIFIC ACTIVITIES OF GLUTAMIC ACID FROM BLOOD AND MILK PROTEIN FRACTIONS FROM A LACTATING RABBIT (NO. X_34, 14TH-17THADAYS POSTPARTUM) INJECTED WITH 150 DC. OF GLUTAMIC ACID—Z-C hrs. post inj. DPM mg. free glutamic acid counted pc./mg. x 10 Glutamic Acid From Blood CY-Globulins 2 22,200 9.200 1.600 6 29,520 7.700 2.540 12 23,570 5.400 2.890 24 18,500 4.900 2.500 36 20,750 6.300 2.180 48 23,580 7.800 2.000 72 19,740 8.900 1.470 Glutamic Acid From Blood /€-Globu1ins 2 28,750 6.400 2.750 6 34,570 4.900 4.670 12 20,380 3.200 4.210 24 25.350 5.500 3.050 36 15,230 5.800 1.740 48 10,680 6.100 1.160 72 4,560 6.300 0.480 TABLE 11. 107 RATIOS OF THE (a) SPECIFIC ACTIVITIES OF GLUTAMIC ACID FROM MILK IMMUNE GLOBULINS TO THE (b) SPECIFIC ACTIVITIES OF GLUTAMIC ACID FROM BLOOD r—GIOBULINS FROM A LACTATING RABBIT (NO. X434, 14TH-17TH DAYS POSTPARTUM) INJECTED WITH 150 pC. OF GLUTAMIC ACID—Z-C hrs. 5 of milk immune globulins post inj . a / b derived from blood 7"- globulins 2 0.917 91.7 6 0.948 94.8 12 0.943 94.3 24 0.889 88.9 36 0 . 918 91. 8 48 0.944 94.4 72 0 . 850 85 . 0 0.916 1 0.01351 lThe mean value for the ratios is presented with standard error. TABLE‘IZ. 108 RATIOS OF THE (a) SPECIFIC ACTIVITIES OF GLUTAMIC ACID FROM "BLOOD" SERUM ALBUMIN FROM MILK TO THE (b) SPECIFIC ACTIVITIES OF GLUTAMIC ACID FROM BLOOD ALBUMIN FROM A LACTATING RABBIT (NO. x_34, lhTH—17TH DAYS EOSTPARTUM) INJECTED WITH 150 p0. OF GLUTAMIC ACID_2.C1 hrs. % of "blood" serum albumin from post inj. a/b milk serum derived from blood albumin 2 0.836 83.6 6 0.935 93.5 12 0.929 92.9 2n 0.937 93.7 36 0.960 96.0 08 0.952 95.2 72 1.033 103.3 0.900 1 0.02161 The mean value of the ratios is presented with standard error. 109 100 90 P5 x Free Blood Glutamic Acid 0 Glutamic Acid From Casein 10.0 /03 ,L/C/mm. H 'o I " \\ \ l 0.10 "’:\L\ l l l l l 1 2h 36 #8 72 hrs. post inj. 0.01 O<‘: N. 0\ F5 FIGURE 29. THE LOG OF SPECIFIC ACTIVITY (um/nun.) vs. TIME (hrs. post inj.) FOR FREE BLOOD GLUTAMIC ACID AND GLUTAMIC ACID FROM CASEIN ISOLATED FROM X—BO (LACTATING RABBIT 14TH-17TH EfiYS POSTPARTUM INJECTED WITH 150. 0 DC. OF DL-GLUTAMIC ACID-2- C 110 100.0 I- X IFree Blood Glutamic Acid Jr 0 Glutamic Acid From 8 -Lactoglobulin 10.0 L— 0 x s‘ £ 1.0 — (3 o :\ x 8” \ O x 0.10 L. . x 0.01 I A l I It I 0 2 6 12 2hr 36 7+8 72 hrs. post inj. FIGURE 30. THE LOG OF THE SPECIFIC ACTIVITY (um/mm.) VS. TIME (hrs. post inj.) FOR FREE BLOOD GLUTAMIC ACID AND GLUTAMIC ACID FROM p-LACTOCIOBULIN ISOLATED FROM x. 30 (LACTATING RABBIT 14TIL17TH DAYS POSTPARTUM “ INJECTED WITH 150.0 p0. OF DL-GLUTAMIC ACID—Z—C ) 111 100.0 .. X Free Blood Glutamic Acid 0 Glutamic Acid From °<-Lacta1bumin 10.0 p x\ 1 A\. o\ x E A 1.. .. 0 C U I i 03 \9 I 0.10 L. ' x’ 0.01 L 4 l L I J .1 I O 2 6 12 2“ V 36 1&8 hrs. post 111.). FIGURE 31. THE LOG 0F SPECIFIC ACTIVITY (um/mm.) vs. TIME (hrs. poet inj.) FOR FREE BLOOD GLUTAMIC ACID AND GLUTAMIC ACID FROM oc-LACTALBUMIN ISOLATED FROM L30 (LACTATING RABBIT lbw-17TH YS POSTPARTUM INJECTED VITH 150.0 110. OF DID-GLUTAMIC ACID-Z- ). 112 100.0 _. x Free Blood Glutamic Acid .1 o Glutamic Acid From Blood Y-Globulins 10.0 A. x\ x g' x E \. u 1 1.0 . 0') x Q \ u 0‘. \. \. ‘ O \ 0.10 L O x 0.01 I I l l I ' ' 0 2 6 12 21+ 36 1+8 '72 hrs. poat 1113. FIGURE 32. THE L00 OF SPECIFIC ACTIVITY (ue./mm.) vs. TIME (hrs. post inj.) FOR FREE BLOOD GLUTAMIC ACID AND GLUTAMIC ACID FROM BLOOD Y-GLOBULINS ISOLATED FROM x. 30 (LACTATING RABBIT RTE-171‘? DAYS POSTPARTUM INJECTED wITR 150.0 p0. 0F DLGLUTAMIC ACID.2-C h). 113 100.0 F. X Free Blood Glutamic Acid . Glutamic Acid From Milk Immune Globulins 10.0 r- E. x E \. U .0 __ 3‘ l g) 7 \ fl .\.\ \ \. I 0.10 - \O 0.01“ A 1 L I l l I 0 2 6 12 2h 36 48 72 hrs. post ind. FIGURE 33. THE 100 OF SPECIFIC ACTIvITY (uc./mm.) vs. TIME (hrs. post. inj.) FOR FREE BLOOD GLUTAMIC ACID AND GLUTAMIC ACID FROM MILK mm GLOBULINS ISOLATED FROM x. 30 (LACTATING RABBIT 1am. 17TH MES POSTPARTUM INJECTED wITB 150.0 NO. OF DL-GLU'I‘AMIC ACID—2-01 ). 114 x Glutamic Acid From Blood Y’ -Globulins 0 Glutamic Acid From Milk Immune Globulins /09 ,uc /mm. 0 f... O I a): / J 1 J l l l I 2 6 .12 2A 36 as 72 hrs. post inj. FIGURE 3h. THE LOG OF THE SPECIFIC ACTIVITY (um/mm.) 0F GLUTAMIC ACID FROM BLOOD Y-GLOBULINS AND MILK IMMUNE GLOBULINS ISOLATED FROM X-BO (LACTATING RABBIT 1bTH-17TH RAYS POSTPARTUM INJECTED WITH 150.0 130. OF DLGLUTAMIC ACID.2.Cl ). 115 l.00---------—--- --—— ----—-—- —--- --__.__ /C\f : = - A e ' V_—. 0.75 .— 0.50 r- 0.25 .— o.oo I l I A I I I 0 2 6 12 2’4 36 1+8 72 hrs. post inj. FIGURE 35. RATIOS OF THE SPECIFIC ACTIVITIES OF GLUTAMIC ACID FROM MILK IMMUNE GLOBULINS TO THE SPECIFIC ACTIVITIES OF GLUTAMIC ACID FROM BLOOD Y -GLOBULINS FROM X-3O (LACTATING RABBIT 14TH-1ZTH DAYS POSTPARTUM INJECTED WITH 150 DC. DL-GLUTAMIC ACID-Z-C 1"" . 116 100.0 _ X Free Blood Glutamic Acid r 1 o Glutamic Acid From Blood Albumin 10.0 '- zflqg /Jc./anrn H 'o T r/\\\> 0.10 '- l l i l J an 36 #8 72 hrs. post inj. NP 0‘— $3 0.01 Or‘ FIGURE 36. THE LOG 0F SPECIFIC ACTIVITY (uc./mm.) VS. TIME (hrs. post inj.) FOR FREE BLOOD GLUTAMIC ACID AND GLUTAMIC ACID FROM BLOOD ALBUMIN ISOLATED FROM x-30 (LACTATING RABBIT lhTH-17TH DAY?“ POSTPARTUM INJECTED'WITH 150.01DC. 0F DL.GLUTAMIC ACID-Z-C ). 100.0 .- l0.0 -— 0.10 "' /03 ,uc./mm. H “o I 117 x Free Blood Glutamic Acid 0 Glutamic Acid From "Blood" Serum Albumin From Milk 0.01 O——: FIGURE 37. l I l l _l l 2 6 12 24 36 48 72 hrs. post inj. THE LOG OF SPECIFIC ACTIVITY (um/mm.) VS. TIME (hrs. post inj.) FOR FREE BLOOD GLUTAMIC ACID AND GLUTAMIC ACID FROM "BLOOD” SERUM ALBUMIN FROM MILK ISOLATED FROM X-30 (LACTATING RABBIT luTH.17T§uDAYS POSTPARTUM INJECTED WITH 150 . 0 DC . 0F DL-GLUTAMIC ACID.2-C ). ~Xo 118 1.0 '- X Glutamic Acid From Blood Albumin ' Glutamic Acid From "Blood" Serum Albumin From Milk /o_9 :c. /mm. ._t<\ / /‘ o 1L #- L. 0.01 I I 20 36 1+8 72 — _ CL---===;_I N- Y3 hrs. post inj. FIGURE 38. THE LOG OF THE SPECIFIC ACTIVITY (um/mm.) VS. TIME (hrs. post inj.) FOR GLUTAMIC ACID FROM BLOOD ALBUMIN AND "BLOOD" SERUM ALBUMIN FROM MILK ISOLATED FROM x—30 (LACTATING RABBIT lhTH-lZTH DAYS POSTPARTUM INJECTED WITH 150.0 130. OF DL-GLUTAMIC ACID-Z-Cl ). 119 1000 F- -------------------------- _ - o/. ' ./ \ / 0.75 - ./ . 0.50 V— 0.00 _L l I l I I l 0 2 6 12 2hr 36 1+8 72 hrs. post inj. F IGURE 39o RATIOS OF THE SPECIFIC ACTIVITIES OF GLUTAI‘CEC ACID FROM "BLOOD" SERUM ALBUMIN FROM MILK TO THE SPECIFIC ACTIVITIES OF GLUTAMIC ACID FROM BLOOD ALBUMIN ISOLATED FROM X-30 (LACTATING RABBIT 14TH-17 ACID-Z- ). TIHDAYS POSTPARTUM INJECTED'WITH 150.0 DC. OF DL-GLUTAMIC C 120 100.0 _. X Free Blood Glutamic Acid ‘ . Glutamic Acid From Blood a{-Globulins 10.0 — x\ x E‘ A \. i 1.0 '— g3 x \ . x O/ \ / .\. O I x 001 '- C X 0.01V I I I I I I I 0 2 12 24 36 #8 72 hrs. pCSt 1113. FIGURE 1+0. THE LOG OF SPECIFIC ACTIVITY (um/mm.) VS. TIME (hrs. post inj.) FOR FREE BLOOD GLUTAMIC ACID AND GLUTAMIC ACID FROM BLOOD °<.GL0BULINS ISOLATED FROM x-30 (LACTATING RABBIT lam-17 “DAYS POSTPARTUM INJECTED WITH 150.0 DC. OF DL.GLUTAMIC ACID—2-0 ) 121 lOOoOF x Free Blood Glutamic Acid Jr 0 Glutamic Acid From Blood B -Globu1_ins 10.0 - E‘ A A. u 3‘ 1.0 p- g1 x \ /\ \ A -\ \ \ x 0.10 - \. X 0.01-V I A L I I I I 0 2 6 12 2L» 36 "#8 72 hrs. post inj. FIGURE #1. LOG 0F SPECIFIC ACTIVITY (um/mm.) VS. TDIE (hrs. post inj.) FOR . FREE BLOOD GLUTAMIC ACID AND GLUTAMIC ACID FROM BLOOD 3 -GLOBULINS ISOLATED FROM x. 30 (LACTATING RABBIT 1hTHa17‘I‘H I IS POSTPARTUM INJECTED WITH 150.0 p0,.-OF DLGIIITAMIC ACID—Z—C ). 122 100.0 r- X Free Blood Glutamic Acid 4, o Glutamic Acid From Casein 10.0 I—\ I x§ R' S - <3 3‘ 1.0 _ “ 3‘ \ ‘\ X I \Y‘ 0.1 "" . x 0001 v I L I I I I I 0 2 6 12 2h ‘ 36 I+8 72 hrs. 130313 1113. FIGURE 142. TREIOG OF SPECIFIC ACTIVITY (um/mm.) VS. TIME (hrs. post inj.) FOR FREE BLOOD GLUTAMIC ACID AND GLUTAMIC ACID FROM CASEIN ISOLATED FROM L30 (LACTATING RABBIT lhTH-17TH DAYS POSTPARTUM INJECTED WITH 150.0 DC. OF DL-GIU'I‘AMC ACID—2-01 ). 123 10000 - X Free Blood Glutamic Acid .r O Glutamic Acid From 8 -Lactoglobulin 10.0 — f ;\ E \. 0 K 1.0 I" * O h \ 0 ‘\ o x‘\“~\“‘\‘\‘ \\\VL\\\\O x 0.1 " I \1\ x' 0.01 I I I I I I J 0 2 6 12 2A 36 1+8 72 hrs. post inj. FIGURE A3. THE L0G OF SPECIFIC ACTIVITY (um/mm.) VS. TIME (hrs. post inj.) FOR FREE BLOOD GLUTAMIC ACID AND GLUTAMIC ACID FROM 3 -LACTOGLOBULIN ISOLATED FROM L31; (LACTATING RABBIT luTH-17T§I DAYS POSTPARTUM IN- JECTED WITH 150.0 p0. 0F DL-GLUTAMIC ACID-Z—C ‘0. 121+ 100.0 .- X Free Blood Glutamic Acid Alp 0 Glutamic Acid From \ OC—Lactalbumin 10.0 i- x o\‘ /03 yen/mm. H I: I x O \1‘ O 0.1 In X 0.01! I I I I I fl I 0 2 6 12 2A 36 » A8 72 _ : hrs. post inj. FIGURE ALI. THE LOG OF SPECIFIC ACTIVITY (uc./mm.) Vs. TIME (hrs. post inj.) FOR FREE BLOOD GLUTAMIC ACID AND GLUTAMIC ACID FROM o<-IACTALBUMIN ISOLATED FROM X-3LI- (LACTATING RABBIT llITH-17TH DAYS POSTPARTUM INJECTED WITH 150.0 DC. 0F DL—GLUTAMIC ACID-Z—C . 125 100.0 -- X Free Blood Glutamic Acid u o Glutamic Acid From Blood 7 —Globulins 10.0 — X\\\\\\ x E \. Q 00 II- x 3‘ 1 U) \3 K .‘o \. \ \. . \ l O 0.1 - X’ 0.01 7 I I I- I I I J 0 2 6 12 2h 36 #8 72 hrs. post inj. FIGURE 45. THE LOG OF SPECIFIC ACTIVITY (um/mm.) VS. TIME (hrs. post inj.) FOR FREE BLOOD GLUTAMIC ACID AND GLUTAMIC ACID FROM BLOOD Y.GLOBULINS ISOLATED FROM L34 (LACTATING RABBIT 14TH.1 “DAYS POSTPARTUM INJECTED WITH 150.0 DC. OF DL-GLUTAMIC ACID.2-C ). 126 100.0 - X Free Blood Glutamic Acid II- 0 Glutamic Acid From Milk Immune Globulins 1090 F- x x\ I i E \ U i 100 - X 0‘3 0 \ fl 0‘0 \. \j \. 4. \ I 0 X ., . o 0.01_ I I I I A I I 0 2 6 12 21+ 36 ‘48 72 hrs. p081; inj. FIGURE 46. THE LOG 0F SPECIFIC ACTIVITY (uc./mm.) vs. TIME (hrs. post inj.) FOR FREE BLOOD GLUTAMIC ACID AND GLUTAMIC ACID FROM MILK IMMUNE GLOBULINS ISOLATED FROM L34 (LACTATING RABBIT 14TH-17TR DAES POSTPARTUM INJECTED WITH 150.0 p0. OF DLGLUTAMIC ACID-2.01 . 127 1.00 '- X Glutamic Acid From Blood '2' -Globulins C Glutamic Acid From Milk ,5‘! Immune Globulins L ALL .1 J I 6 12 29 36 48 72 hrs a pOSt inj 0 FIGURE 47. THE LOG OF SPECIFIC ACTIVITY (uc./mm.) vs. TIME (hrs. post inj.) FOR GLUTAMIC ACID FROM BLOOD ‘r—GLOBULINS AND MILK IMMUNE GLOBULINS ISOLATED FROM X-314- (LACTATING RABBIT lhTH-17TH DAES POSTPARTUM INJECTED WITH 150.0 DC. OF DL-GLUTAMIC ACID-Z-C ). 128 1.00r-------. ---------------------- . ______ /.—- #:f - . fi’i \K. 0.75 L 0.50 P 0.25 - 0.00 l l I I g I I 0 2 6 12 24 36 48 72 hrs. post inj. FIGURE #8. RATIOS OF THE SPECIFIC ACTIVITIES 0F GLUTAMIC ACID. FROM MILK IMMUNE GLOBULINS TO THE SPECIFIC ACTIVITIES 0F GLUTAMIC ACID FROM BLOOD ‘T-GLOBULINS ISOLATED FROM L34 (LACTATING RABBIT 14TH-17TH DAYS POSTEARTUM INJECTED WITH 150.0 p0. OF DL- GLUTAMIC ACIDuZ-Cl . 129 100.0 — 1! Free Blood Glutamic Acid Ir 0 Glutamic Acid From Blood Albumin 10.0 P /03 [Jo/mm. '3' I . x I 0.1 -— x\x \.\ .\: #- I I I I 12 21+ 36 1+8 72 hrs. post inj. 0.01% I i 02 6 FIGURE 49. THE LOG OF THE SPECIFIC ACTIVITY (uc./mm.) VS. TIME (hrs. post inj.) FOR FREE BLOOD GLUTAMIC ACID AND GLUTAMIC ACID FROM BLOOD ALBUMIN ISOLATED FROM L34 (LACTATING RABBIT 14TH-17TH DAYS POSTPARTUM INJECTED WITH 150.0 NO. OF DL—GLUTAMIC ACID-Z—C ) 130 100.0 .— X Free Blood Glutamic Acid 1: e Glutamic Acid FromFBloOd" Serum Albumin From mlk 10.0 P" \, \. ./ \.\. ‘ X. /03 ,uc./mm. f“ O I I I I I I I 24 36 #8 72 hrs. post “.30 0.01 0" ~— F5 FIGURE 50. THE LOG 0F SPECIFIC ACTIVITY (um/mm.) vs. TIME (hreypoet inj.) FOR FREE BLOOD GLUTAMIC ACID AND GLUTAMIC ACID FROM "BLOOD" SERUM ALBUMIN FROM MILK ISOLATED FROM L34 (LACTATING RABBIT 14TL17TH DAYS POSTPARTUM. INJECTED WITH 150.0 130. OF DLGLUTAMIC ACID—2-01“). 131 1.00 .— x Glutamic Acid From Blood Albumin ' Glutamic Acid From "Blood” Serum Albumin From Milk 0 O [.1 O I / X /03 ,uc /mm x/ I I I I I I 214 36 ’48 72 0.01 0“: NI- 0\ 5 hrs . post inj. FIGURE 51. THE LOG OF SPECIFIC ACTIVITY (uc./mm.) VS. TIME (hrs. post inj.) FOR GLUTAMIC ACID FROM BLOOD ALBUMIN AND "BLOOD" SERUM ALBUMIN FROM MILK ISOLATED FROM L34 (LACTATING RABBIT 14TH.17TH DA S POSTPARTUM INJECTED WITH 150.0 DC. 0F DLGLUTAMIC ACID—2.0l ). 132 1°°°"‘“.:__"‘.“““". ““““ . ''''''' :_.—+———----- O/ 007.5 "’ 0.50 .— 0.25L‘ 0.00 I I I I I I J 0 2 6 12 24 36 48 72 hrs. post inj. FIGURE 52. RATIOS OF THE SPECIFIC ACTIVITIES OF GLUTAMIC ACID FROM ”BLOOD" SERUM ALBUMIN FROM MILK TO THE SPECIFIC ACTIVITIES 0F GLUTAMIC ACID FROM BLOOD ALBUMIN ISOLATED FROM L34 (LACTATING RABBIT 14TH-17 DAYS POSTPARTUM INJECTED WITH 150.0 p0. OF DLGLUTAMIC ACID.2-C L’) . 133 100.0 p X Free Blood Glutamic Acid Jr 0 Glutamic Acid From Blood \\ OC-Globulins 1000 "x X\ I E \ d 1.0 " x 3. G") e \ . X ./ \. / \I;. o N r O 0.10 b X 0.01 _ I l L is ' ' ' ‘0 2 6 12 2h 36 #8 72 hrs. post inj. FIGURE 53. THE LOG OF THE SPECIFIC ACTIVITY (um/mm.) VS. TIME (hrs. post inj.) FOR FREE BLOOD GLUTAMIC ACID AND GLUTAMIC ACID FROM BLOOD °<—GIDBULINS ISOLATED FROM L34 (LACTATING RABBIT 14TH.17TH DAYS POSTPARTUM INJECTED WITH 150.0 p0. OF DL-GLUTAMIC ACID—2-01“). 134 100.0 — )( FIee Blood Glutamic Acid Jr 0 Glutamic Acid From Blood B -Globulins 10.0 "—x X\ x E \. 1.0 r- x U 3A 01 o \ .\. / \ X 0 I \ O \. 0.10 I- O O O O H - - - I 02 6 12 24 36 148 72 hrs. post inj. FIGURE 54. THE LOG OF SPECIFIC ACTIVITY (um/mm.) VS. TIME (hrs. post inj.) FOR FREE BLOOD GLUTAMIC ACID AND GLUTAMIC ACID FROM BLOOD £-GLOBULINS ISOLATED FROM L34 (LACTATING RABBIT 14TH.17TH DAYS POSTPARTUM INJECTED WITH 150.0 pC. OF DL-GLUTAMIC ACID—2-01“). 135 Preliminary work indicated that an injection of 50 pc. of glutamic “014 acid~2 was too low to give sufficient labeling in the milk proteins of lactating rabbits. The levels of activity were so low that the data could not be evaluated. This is the reason that such high levels of activity were injected into these animals. The extremely rapid decline in the specific activity of free blood glutamic acid can be attributed to an extremely rapid metabolism of glutamic acid in the rabbit. This point should also be considered along with the other points discusSed earlier for the rapid decline in specific activity of free blood leucine. There are no other data available on the incorporation of glutamic acid into milk proteins by the rabbit. These data indicate that the lactating rabbit incorporates a greater percentage of free blood glu- tamic acid into caseins and.93«lactcglobulin than the goat (Barry, 1958). There are no other data available indicating that the glutamic acid of”0has stated that cows injected with antigens of various kinds directly into the mammary gland via the teat will produce specific antibodies within the milk. ;Also, Askonas and Humphrey (1958) have shown local production of antibodies when a tissue is injected with an antigen. However, these data and the results with labeled leucine and glutamic acid provide good evidence that the normal, healthy mammary gland does not produce the immune globulins of milk. It just transfers them from.the interstitial fluid pool to the milk. Smith (1948) reported that the percent of leucine in the immune globulins of cow's milk is about 20% higher than in the'7’-globulins of 1% the blood. This would mean an incorporation of 2 additional mg. of leucine per 100 mg. of milk immune globulins by the mammary tissue. This would cause a dilution of the specific activity (pm/mg. of protein) of the milk immune globulins and thus yield values different from the specific activities of blood Y-globulins. The work of Larson and Gillespie (1957) and Larson (1958) has shown that fiz-globulins are also transferred to the immune globulins of cow's milk. The findings of Smith (19148), Larson and Gillespie (1957) and Larson (1958) do not appear to be true in the case of these two rabbits. It seems that the specific activities of the immune globulins of the milk of these two animals would have been different from the specific activities of the Y-globulins of the blood if the non-labeled flz-globulins and addi- tional non-labeled leucine had been diluting the immune globulins in the rabbits' milk. There appears to be a species difference between the rabbit and cow. Fink e1 33,. (191414) were the first to inject labeled proteins into the living animal to study their behavior in 31112. Since that time, many studies have been done using 1131, Cl“ and N15 labeled plasma proteins and purified proteins to study their distribution in body fluids, turnover rates, and biological half-times. Dixon gt 3],. (1952), Dovey at 51. (1951+), Germuth at al. (1951), and Cohen at al. (1956) have reported half-times for T‘ -globulins injected into rabbitsmanging from 3.2 to 5.7 days. Inspection of the graphs for the specific activ- ity of blood T-globulins in Figures 55 and 56 reveals a half-time of 2 to 3 days as determined by the graphic method. This is not unusual, since the Y-globulins were leaving the body via the mammary secretions in this case. The data of the investigators above were reported on non- lactating rabbits. The slope of these curves is greater during the 1&7 first few hours post injection. This is due to equilibration of the Clu-YC-globulin'with the tissue fluids. ‘Wasserman and Meyerson (1951) reported that equilibration of injected 1131 labeled albumin required 7 hours for complete equilibration into the lymph and interstitial fluid in the dog. It appears that the equilibration of‘yfi-globulin in the rabbit was a little faster. The work presented in Tables 13 and 1h is the only example of homologous Clu-jfsglobulin injected intravenouSly into lactating rabbits and the subsequent isolation of immune globulins of milk with the same specific activity. On the basis of this study and the results presented earlier, it has been shown that the immune globulins of rabbit milk are derived directly from the‘Y’-globulins of the blood. These conclusions are in agreement with conclusions reached from studies on the cow. Additional data on the specific activities of various blood and Ola-W’-globulin are presented milk protein fractions after injection of in Tables 6 and 77 in the appendix. The activity in these fractions is very low and of little significance. It appears to be due to amino acids released from the catabolism of the injected radioactive‘1’- globulin o 148 D. EXperiments 7 and 8. Experiments Designed to Demonstrate the Transfer of Blood Albumins to Mammary Secretions of the Rabbit Since the experiments with Clu47'-globulin demonstrated clearly the transfer of this protein to mammary secretions, the experiment was repeated using Clusalbumins. Tables 17 and 18 and Figures 59 and 60 present the Specific activities of blood albumins and "blood" serum albumins from milk of the two lactating rabbits used in these experi— ments. These data show that after equilibration with the interstitial fluids, there is remarkable agreement between the specific activities of the blood albumin and the "blood" serum albumin in milk. In these experiments, the equilibration time appears to be somewhere between 6 and 12 hours for both animals. This is a little longer than in the case of the injected'quglobulins. This is interesting in view of the fact that the molecular weight of albumin is 60,000 to 70,000 and the molecular weight of the bloodTY'-globulin is around 160,000. It should be remembered that the negative charge on blood albumin is greater than the charge on'y'uglobulin, since the mobility of albumin is much greater than that of 1’-globulin during electrophoresis at pH 8.6. This might explain some of the difference in the time of equilibration. It is postulated that there is some mechanism in the epithelial cells of the mammary gland which is responsible for the above difference. This is not only evident because of the time relationship but alSo be- cause of the quantitative differences. The albumin fraction of blood is considerably larger than the'Y'-globulin fraction of blood. The re- verse is true in the milk. The immune globulin fraction of the milk is much greater than the 'blood" serum albumin fraction. If these two pro- teins were entering the mammary secretions by the same mechanism, it 149 TABLE 17. THE SPECIFIC ACTIVITIES OF BLOOD ALBUMIN AND "BLOOD” SERUM ALBUMIN FROM MILK FROM A LACTATING RABBIT (NO. X-17, lOT'gE _ C 13TH DAYS POSTPARTUM) INJECTED'WITH 5.16‘pC. (2.0 gm.) LABELED ALBUMIN. posirinj. DPM 'mg. protein counted ‘pc./mg. protein x 10“ Blood Albumin 0.16 12,480 40.0 1.47 2 13,000 44.9 1.30 6 11,835 47.8 1.12 12 10,400 46.3 1.02 24 9,348 48.1 0.88 48 6,335 42.5 0.67 72 5,340 49.0 0.47 "Blood" Serum Albumin From Milk Serum 2 615.0 0.34 0.82 6 404.0 0.18 1.00 12 518.0 0.24 0.98 24 429.5 0.27 0.72 48 620.0 0.40 0.70 72 12.01 0.12 0.45 l The count of this sample was not significantly different from background. TABLE 18. 150 THE SPECIFIC ACTIVITIES OF BLOOD ALBUMIN AND "BLOOD” SERUM ALBUMIN FROM MILK FROM A LACTATING RABBIT (NO. X-ZO, 10TH~ 1EEH DAYS POSTPARTUM) INJECTED'WITH 13.2 p0. (3.0 gm.) C LABELED ALBUMIN. posgrinj. DPM mg. protein counted pc./mg. protein x 10'!+ 1‘ Blood Albumin 0.16 22,695 33.6 3.05 2 29,250 50.2 2.65 6 24,795 48.5 2.30 12 19,915 41.6 2.15 24 19,505 45.0 1.95 48 15,600 39.3 1.79 72 14.990 47.0 1.35 "Blood” Serum Albumin From.Milk Serum 2 140.0 0.40 1.58 6 83.5 0.21 1.79 12 126.0 0.27 2.11 24 63.0 0.15 1.94 48 148.1 0.38 1.76 72 35.2 0.12 1.32 151 10.0 r. x Blood Albumin 0 ”Blood” Serum Albumin V' From Milk 52 x 6: ‘L\* E. o —- t i ./ §X\ .¥ ‘5' g) \ ‘\ 0.1 11 1 1 1 1 1 I 2 6 12 24 36 48 72 hrs. post inj. FIGURE 59. THE LOG OF THE SPECIFIC ACTIVITY (uc./m8. x 10“) vs. TIME (hrs. post inj.) FOR BLOOD ALBUMIN AND ”BLOOD" SERUM ALBUMIN FROM MILK ISOLATED FROM x-l7 (LACTATING RABB 10TH-13TH DAYS POSTPARTUM INJECTED mm 5.16 DC. OF C LABELED ALBUMIN). Lt 152 10.0 ’ 8 Blood Albumin ° "Blood" Serum Albumin l\h From Milk V~ x ' Q \x\ \ $— " /°/ fi" —* s \ — d 1.0 3. 0') O \ 0.1 I 1 i ' ‘ ' ‘ 0 2 6 12 24 36 48 72 hrs. post inj. FIGURE 60. THE‘LOG OF THE SPECIFIC ACTIVITY (uc./mg. x 10“) vs. TIME (hrs. post inj.) FOR BLOOD ALBUMIN AND “BLOOD" SERUM ALBUMIN FROM MILK ISOLATED FROM X-20 (LACTATING RABB T 10TH..l3TH DAYS POSTPARTUM INJECTED WITH 13.2 DC. OF Cl LABELED ALBUMIN). 153 seems highly probable that the ratio of albumin to globulin would be the Asame in both blood and milk. This definitely is not the case. Therefore, it is believed that the mammary tissue actively takes up the blood 7‘— globulins or that they pass from the blood to the mammary secretion with relative ease. Due to the extremely low concentration of "blood" serum albumin in milk, it appears that the blood albumin enters the mammary secretion in a passive manner. This could possibly be due to a hydro- static pressure phenomena. Another possibility is that the albumin is passed from the interstitial fluid through the mammary epithelium with difficulty. This might be due to the charge on the albumin molecule. These conclusions on the incorporation of blood albumin in mammary secretions are supported by the recent work of Lecce and Legates (1959). They have reported that the levels of blood serum albumin and immune globulins increase in the cow’s milk during mastitis. This may indicate a greater permeability to albumin by the mammary epithelium. Tables 19 and 20 and Figures 61 and 62 present the ratios of the specific activities of "blood" serum albumin in milk to the specific activities of blood albumin. It is obvious that after 12 hours there is a high degree of similarity between the two protein fractions. From the means of these ratios, it can be calculated that 92.2% of the "blood" serum albumin in milk in one animal and 98.2% of the "blood" serum albumin in milk in the other animal was derived from the blood albumin after the first 12 hours post injection. Additional data on the specific activities of other blood and milk fractions from these animals are presented in Tables 8 and 9 in the appendix. The activity in these fractions is very low and of little significance. It is due to amino acids released by the catabolism of the injected radioactive albumin. TABLE 19. 154 THE RATIOS OF THE (a) SPECIFIC ACTIVITIES OF "BLOOD" SERUM ALBUMIN FROM MILK TO THE (b) SPECIFIC ACTIVITIES OF BLOOD ALBUMIN FROM A LACTATING RABBIT (NO. X—17, 10 -13TH DAYS POSTPARTUM) INJECTED WITH 5.16‘pC (2.0 gm.) 01 LABELED ALBUMIN. hrs. % of "blood" serum albumin from post inj. a/b milk derived from blood serum albumin 2 0.557 -__ 6 0.777 --- 12 0.962 96.2 24 0.819 81.9 48 1.040 100.0 72 0.958 95.8 0.970 t 0.0482l 1The mean value of the ratios from 12 through 72 hours post injection with standard error. TABLE 20. 155 THE RATIOS OF THE (a) SPECIFIC ACTIVITIES OF "BLOOD" SERUM .ALBUMIN FROM MILK TO THE (b) SPECIFIC ACTIVITIES OF BLOOD ALBUMIN FROM A LACTATING RABBIT (NO. x-20, 10TH-13TH DAYS POSTPARTUM) INJECTED'WITH 13.2 US. (3.0 gm.) Cl“ LABELED ALBUMIN. hrs. 6 of "blood" serum albumin from post inj. a/b milk derived from blood albumin 2 0.518 --_ 6 0.675 . -_- 12 0.982 98.2 24 0.995 99.5 48 0.984 98.4 72 0.978 97.8 0.985 1 0.003211 1The mean value of the ratios from 12 through 72 hours post injection is presented with standard error. 156 1.00 -— ———————————————————— 7"»: 0075 - /. 0.50 r- 0.25 I— 0.00 l I I I I l I O 2 6 12 24 36 48 72 hrs. post inj. FIGURE 61. RATIOS OF THE SPECIFIC ACTIVITIES OF "BLOOD" SERUM ALBUMINs FROM MILK TO THE SPECIFIC ACTIVITIES OF BLOOD ALBUMIN FROM x-l7 (LACTATING RAB IT 10TH- 13TH DAYS POSTPARTUM INJECTED WITH 5.16 no. OF C1 LABELED ALBUMIN). 157 1.00 -—------.- —————— - ------ 1---—--._.\--,__--.- 0.75 *- / 0.50 - . 0.25 F- 0,00 1 1 1 I l l I 0 2 6 12 24 36 48 72 hrs. post inj. FIGURE 62. RATIOS OF THE SPECIFIC ACTIVITIES OF ”BLOOD" SERUM ALBUMINS FROM MILK TO THE SPECIFIC ACTIVITIES OF BLOOD ALBUMIN FROM x..2o (LACTATING RAB IT 10TH.13TH DAYS POSTPARTUM INJECTED WITH 13.2 110. OF Cl LABELED ALBUMIN). 158 There have been many reports on turnover rate and biological half- times of serum albumins in various species. Dixon.gt,a1. (1953) and Cohen et a1. (1956) have reported half-time values for radioactive alu bumins injected into rabbits as 5.7 and 3.8 days, respectively. These reported values agree fairly well with the half-times determined by inspection of the specific activity curves for blood albumins presented in Figures 59 and 69, which are approximately 3 days. These data presented in Tables 17 through 20 are interesting, since they provide new information concerning albumin in rabbit milk. ”Blood” serum albumin in rabbit milk has not been reported in the literature. This is the first work on the injection of Cln-allmmin into a lactating animal 0 159 E. EXperiments 9 and 10. Experiments Designed to Evaluate the Potential ofo<~Globulins and ,8 ~uGlobulins as Precursors of Rabbit Milk Proteins The possibility of c( -- and fl ~globulins as precursors of milk prom teins produced by the rabbit was also investigated. The data for these experiments are presented in Tables 10 and 11 in the appendix. All of the blood and milk protein fractions mentioned previously were isolated and the Specific activity of these fractions determined. It can be seen that the activities in all of these fractions were much lower than the activity in the injected fraction. On the basis of these data, it may be concluded that o<~ and fl -blood globulins do not serve as direct precursors of milk proteins nor are any of the milk proteins isolated derived directly from them. The activity which does appear is attrie buted to amino acids liberated in the body from the catabolism of these radioactive injected fractions. These data do not preclude the possibility of milk proteins which are derived directly from o(- and p -g1obulins. From the conclusions drawn from.the work done on albumins, it seems reasonable to postulate that all the proteins of blood plasma are present in minute quantities Vin milk. However, it is felt that they are present in such minute quantities that present qualitative methods are not sensitive enough to detect them. This is not unreasonable when the protein hormones are considered. It is certain that they are present in the blood but they have not been chemically isolated and characterized. It would be even more difficult to detect the minor blood proteins in milk since they do not.have the physiological activity of the various hormones. II. Studies of the Hormonal Requirements of Guinea Pig Mammary Tissue In Eliza ' A. Results 1. Dexelameni. ms.’ 9111mm Me dime. The original plan for the tissue culture work contained three basic ideas: (1) to deve10p a medium capable of maintaining mammary tissue in72132g, (2) to deve10p a medium capable of initiating secretion in non- secretory mammary tissue in_yitzg and (3) to deve10p a medium capable of maintaining secretion of mammary tissue in_yitzg. Two of these aims have been accomplished. Media have been deve10ped that will maintain non-secretory and secretory mammary tissue from guinea pigs in,y11zg. Tables 21 and 22 present the media developed. Table 23 presents a medium prepared as a control to demonstrate the effects of the hormOnes added to the medium. Parker's 199 synthetic medium is a completely synthetic solution composed of 81 nutrients. It is composed of all the known essentials in proper proportions for cell nutrition, containing all of the amino acids, salts, vitamins, and carbohydrates shown to be necessary for cellular maintenance and mitosis. This medium was developed by MOrgan.etHal. (1950). The final concentrations of hormones added to the basic medium are reported in Tables 21 and 22. These are reported in ug./ml. The hydro- cortisone used was from Merck, Sharp, and Dohme (Lot No. 280 0502D) and the prolactin was Panlitar (list 806) Ovine Lactogenic Hormone from Armour Laboratories, Chicago, Illinois. It contained 20 I.U./mg. The insulin preparation used was the crystalline Zn-insulin preparation of Eli Tilly and Co. This preparation contained 25 units of activity per mg. Penicillin.was added as a bacteriostatic. It has been known for a long time that serum added to a tissue culture medium is very beneficial 161 TABLE 21. COMPOSITION OF MEDIUM I DEVELOPED FOR MAINTENANCE OF GUINEA PIG MAMMARY TISSUE IN YIERQ. Parker°s 199 synthetic medium, adjusted to pH 7.4 with 1.4% of NaH003 maisrials.addsd canssniraiicn hydrocortisone 8 ug./m1. prolactin 140 ug./ml. insulin 70 ug./m1. penicillin G 50 I.U./ml. rabbit serum 10% medium.gassed with 95% 02 and 5% CO2 162 TABLE 22. COMPOSITION OF MEDIUM II FOR MAINTENANCE OF ACTIVE SECRETION OF GUINEA PIG MAMMARY TISSUE INLIIEEQ. Parker's 199 synthetic medium, adjusted to pH 7.4 with 1.4% NaHCO3 maiszialsasiisi ssnssnizaiicn hydrocortisone 16 ug./m1. prolactin 240 ug./ml. insulin 140 ug./ml. penicillin G 50 I.U./m1. rabbit serum 10% medium gassed with 95% 02 and 5% CO2 163 TABLE 23. COMPOSITION OF CONTROL MEDIUM FOR GUINEA PIG MAMMARY TISSUE 11.1 111229. Parker°s 199 synthetic medium, adjusted to pH 7.4 with 1.4% NaHCO3 Wad WE penicillin G ‘ 50 I.U./ml. rabbit serum 10% medium gassed with 95% 02 and 5% CO2 164 to the cell and has become a routine procedure in tissue culture laboratories. The control medium reported in Table 23 was prepared specifically for the purpose of demonstrating the difference between tissues cultured in the presence or absence of the hormones. It was prepared in the same manner as the first 2 media described. All the media were adjusted to pH 7.4 with sterile 1.4% NaHCO3. The 199 medium contains methyl red. The NaHCOB is added a drop at a time and the red color deve10ped is compared with a standard methyl red solution. This method causes a slight variation in pH, but this does not seem to be significant. The three media were usually pre- pared in 50 m1. volumes. Two weeks was the maximum time period that these media were kept. After this length of time, the media were dis- carded as a precautionary measure to prevent contamination and variations in the media. Table 24 presents a summary of the reactions of tissues taken from mammary tissue in a non-secretory state and cultured for 5 days in :medium I. .The average data presented at the bottom of Table 24 sump marize the results of all the cultures on non-secretory mammary tissue .from guinea pigs. The tissues were taken from 8 animals. From these 8 animals, a total of 108 explants were prepared and cultured in medium I. Histological examination of these explants revealed that approx- imately'73% of the total lobulo—alveolar tissue was maintained. The secretory alveoli amounted to only 4.7% of the total alveoli in these explants. This is not considered to be significant due to the crude 165 TABLE 24. RESULTS OF EXPLANTS PREPARED FROM NON-SECRETORI GUINEA PIG 'MAMMARY TISSUE AND CULTURED IN MEDIUM I FOR FIVE DAYS. Animal % of providigg No. % if tgtal total LA explt. explt. LA MT secretory Comments . X-4 12 100.0 0.00 LA appeared like the FTC“. X-7 12 25.0 0.00 Most of the explts. were fatty tissue. The LA of FTC appeared to be involuted. X—8 12 87.5 0.00 LA appeared like the FTC. X-9 12 70.0 6.65 Very slight secretion in some peripheral LA. X-15 12 75.0 18.75 Excellent MT of 9 explt. Three 'were completely degenerate. Secretion in periphery. x46 12 62.5- ' 0.00 Degenerate LA in center of 9 explants. Three explants had enlarged LA. X-33 24 87.5 8.75 Degenerate LA in center of 6 explants. Secretory LA in periphery of 9 explants. AVS. 108 73.6 n.7o MT m appeared similar to FTC. The degenerate LA was usually in center of explt. The secretory LA was at periphery of explt. ILA == lobulo-alveolar tissue 2Explt. = explants 3m =maintained “FTC = Fresh tissue control 166 methods for evaluating the percentage of alveoli that are maintained or secretory. The data presented in Table 24 provide information about the individ- ual experiments with tissues taken from individual animals. It can be seen that there is considerable variation from one experiment to the next. The percent of lobulo-alveolar tissue maintained varies from 100 to 25%. This extreme variation is noted in explants from animal number X-4, which was evaluated as 100% of the lobulo-alveolar tissue maintained, and ex- plants from animal number X-7, which was evaluated as only'25% of the lobulowalveolar tissue maintained. The results of the experiments on the eXplants‘from the last 6 animals are more consistent. In these 6 experiments, the percent of lobulo-alveolar tissue maintained varies from 62.5% to 95% of the lobular-alveolar tissue present in the tissue. The comments in the last column of Table 24 give some insight into the morphological condition of the tissue of these explants. Animal X—7, which provided explants that had the lowest percentage of lobulo- alveolar tissue maintained, had mammary tissue which was in a state of involution. This condition might have influenced the in,11§rg,main— tenance of the alveoli. An interesting point is illustrated in the behavior of the eXplants prepared from animal X-15. In this case, 3 of the explants were completely degenerate while the other 9 explants were completely maintained. This is not an uncommon experience in tissue culture. Some general statements can be made concerning the inbzitzg,main- tenance of guinea pig mammary tissue reported in Table 24. The lobulo- ‘alveolar tissue maintained appeared very similar to the lobulo-alveolar tissue of‘the control tissue taken at the same time that the explants 167 were prepared. Where degeneration of the alveoli occurred, it usually appeared within the center and not in the periphery of the explant. Also, secretory alveoli in the explants cultured in medium I were invariably found in the periphery of the explant, in direct contact with the nutrient medium. Figure 63 is a photomicrograph of a non-secretory control guinea pig mammary tissue. There is no secretion evident in this tissue. The alveoli are small. The epithelial cells do not show typical secretory vacuoles and secretory granuoles are not prominent in the cytoplasm. Figure 64 is a photomicrograph of an explant taken from the same animal at the same time and cultured in medium I for 5 days. The histological picture is the same as the histological picture of the control. These two photomicrographs demonstrate the ability of medium I to maintain guinea pig nonmsecretory mammary tissue. Table 25 presents a summary of the data on tissues taken from mammary glands in a secretory state and cultured for 5 days in medium II. The data presented at the end of Table 25 summarize the results of the tissue culture experiments done on secretory guinea pig mammary tissue cultured in medium II. In these experiments, 12 animals were utilized to provide secretory mammary tissue with a total of 180 individual explants. Seventy percent of the lobulo-alveolar tissue was maintained in these 180 explants presented in Table 25. This is in remarkable agreement with the percent of lobulo-alveolar tissue maintained in the experiments presented in Table 24. The value given in Table 24 is 73.6% of the lobulo-alveolar tissue maintained. Hewever, Table 25 168 PHOTOMCROGRAPH (1301) OF NON-SECRETORI CONTROL GUINEA PIG MAMMARY TISSUE SECTION STAINED WITH IRON HEMATOXTLIN AND EOSIN. FIGURE 63. 169 .PHOTOMCROGRAPH (130X) OF A SECTION OF AN EXPLANT PREPAREDFROMTHESAMEANIMALASTHECONTROL TISSUE (FIGURE 63) AND CULTURED IN MEDIUM I FOR FIVE DAYS. FIGURE 64. 170 TABLE 25. RESULTS FROM EXPLANTS PREPARED FROM SECRETORY GUINEA PIG MAM; 'MARY TISSUE AND CULTURED,IN MEDIUM II FOR FIVE DAYS. Animal % of providing No. % 8f tStal total LA explt. explt. LA MT secretory Comments x- 5 12 100.00 100.0 Secretion same as FTC)". X~6 12 8.75 0.0 Most LA degenerate,tissue diam organized. % Explt. contaminated. X-lO 12 0.00 0.0 All explt. degenerate. All cone taminated. X~14 12 50.00 0.0 No secretion in MT LA. X~17 12 46.00 46.0 Secretion same as FTC. X-21 12 75.00 10.0 Secretion same as FTC. Xe22 12 75.00 75.0 Heavy secretion in MT LA. De- generation in center of 6 eXplt. X~23 24 75.00 75.0 Secretion same as FTC. X—25 24 100.00 100.0 Secretion greater than FTC. Xm26 24 73.00 31.3 All cells highly stimulated and vacuolated. Nuclei greater than FTC. FTC. very slightly secretory. X~28 12 100.00 87.5 All cells highly stimulated and vacuolated. Heavy secretion in secretory LA. No secretion in FTC. X—29 12 100.00 100.0 All cells highly stimulated and vacuolated. All LA shows good secretion. Very little secretion in FTC. AVS. 180 70.00 55.4 Secretion in LA of explt. as good or greater than FTC. Cells con— sistently show enlarged nuclei and secretory vacuoles in the cytoplasm. lEbcplt. = explants 21A.= lobulo-alveolar tissue 3MT = maintained “FTC = fresh tissue control 171 presents a value of 55.4% for the lobuloualveolar tissue which was secretory. This is significantly greater than the value of 4.7% re- ported in Table 24. Two of the experiments reported in Table 25 were faulty because of contamination. It is felt that this is the reason that the explants from these two experiments showed extensive degeneration and no secretory activity. If the values for these two experiments are discarded, the percentage of secretory lobulo-alveolar tissue maintained would have been higher. If these values are discarded, the average values at the end of Table 25 would be 80.3% of the total lobulo-alveolar tissue maintained and 64.0% of the total lobulo—alveolar tissue secretory. The values pre_ sented in Table 25 show a large variation from one experiment to the next. The lobulo-alveolar tissue in explants prepared from animal X~17 was 46% maintained. while that from L5. L25, X-28 and x-29 appeared to be 100% maintained. The same variation is evident in the secretory lobulo- alveolar tissue. The lobulo-alveolar tissue in explants prepared from X—21 was only 10% secretory while the lobulo-alveolar tissue in explants prepared from X-5, X—25 and X-29 appeared to be 100% secretory. It is indicated in the last column of Table 25 that in those cultures that were contaminated by bacteria (X-6 and X-lO), extensive degeneration of the explants took place. At the end of Table 25 the morphological condition of the explants is summarized. It is apparent that in most of the explants in which secretion was noted, the secretion was as great or greater than in the control of the fresh tissue taken at the time the explants were prepared. Also, the nuclei of the epithelial cells appear to be enlarged when compared to the controls. In those alveoli showing a heavy secretion in the lumen, the epithelial cells lining it appear to have large secretory vacuoles in the cytOplasm. 172 Figure 65 is a photomicrograph of secretory guinea pig control tissue. The secretion seen in this tissue is of a very low order of magnitude. Secretory mammary tissue was obtained from guinea pigs on the 4th to 6th days posppartum. They were kept with their litters and the litters were allowed to suckle from.birth until the sows were sacrificed to provide explants. All secretory control tissues had this appearance. It is difficult to explain this low level of secretory activity. It is possible that the litters did not sucklew The newborn guinea pig is not dependent on the mother's milk, and can survive well on dry feed. Figure 66 is a photomicrograph of an explant taken from the same animal as the control in Figure 65 and cultured in medium II for 5 days. There is an obvious difference in the appearance of these two tissues from the same animal. The explant shows considerably more secretory activity than the control. The alveoli are enlarged and secretory materials are evident in the alveolar lumina. The nuclei of the epi- thelial cells are enlarged and secretory vacuoles are very evident in the cytOplasm. The large duct on the left side of the photomicrograph appears to be filled with secretion. It is evident by comparison of Figures 65 and 66 that the secretory activity of this tissue was main- tained and likely increased by medium II. Figure 67 is a photomicrograph of an eXplant from the same animal as the control tissue in Figure 65. It was cultured in the control medium (Table 23) for 5 days. This photomicrograph is a dramatic demonstration of the hormonal requirements of guinea pig mammary tissue 12.11329. This explant is completely degenerate. There do not appear to be any viable cells in this explant. It cannot be recognized as mammary tissue. 173 ’l/ b ( ' "4o." v : » . o ‘..' .59.. \‘g.’ . \ ~.. -$ u-l a“ '. c... ':' . ~ L r FIGURE 65. PHOTOMICROGRAPH (130]!) OF A SECRETORY CONTROL MAMMARY TISSUE TAKEN FROM A LACTATING GUINEA PIG.STAINED WITH HEMATOXYLIN AND mSIN. 174 PHOTOLECROGRAPH (130X) OF A SECTION OF AN EXPIAN‘I‘ FIGURE 66. TAKENFROMTIIESAMETISSUESHWNINFIGURE65AND CULTURED FORFIVEDAISINMEDIUM II. 175 FIGURE 67. PHOTOMICROGRAPH (1301) OF A SECTION OF AN EXPLANT PREPARED FROM THE SAME TISSUE AS SHOJN IN FIGURE 65 AND CULTURED FOR FIVE DAYS IN THE CONTROL MEDIUM. 176 There is a possibility that the apparent secretion observed in the photomicrograph in Figure 66 is an artifact and not the accumulation of true secretory prodtcts. In order to establish that the mammary tissue explants were actually taking up materials from the medium and secreting them into the alveoli via the epithelial cells.0.2 pc. of either DL- leuc“me—2.01;p or Dleglutamic acid-2eclu'was added to medium II. Figures 68 and 69 are photomicrographs of autoradiographs prepared from eXplants cultured in radioactive mediums. Figure 68 is from an explant cultured in a,medium.containing the labeled glutamic acid and Figure 69 is from an explant cultured in a medium containing the labeled leucine. It may be seen from these autoradiographs that these labeled amino acids were taken up and concentrated in certain areas of the tissue. Photomicrographs of autoradiographs are difficult to prepare. In order to show the exposed developed emulsion, the Optical system of the microscope must be focused on the emulsion spread over the tissue. Be- cause of this, the tissue is out of focus. ‘When the slide is placed under the microsc0pe, it is possible to focus on the emulsion and then refocus the same field on the stained tissue. By this technique, it was possible to determine that the areas of concentrated radioactivity were located directly over highly secretory alveoli. This provides further evidence that these explants cultured in medium II were able to take up these radioactive amino acids and concentrate them in the areas of active secretion. It seems reasonable to expect that these amino acids were being incorporated into mammary secretory materials.‘ One autoradiograph showed exposed emulsion along the entire length of a duct which was cut in longitudinal section. Unfortunately, the degree 177 FIGURE 68. PHOTOIECROGRAPH 01? AN AUTORADIOGRAPH PREPARED FROM AN mommroammrsm IUMJICONTAINING 0.2 pc./ml. OF DL-GLUTAMIC ACID—2-01 . FIGURE 69. PHOTOMICROGRAPH OF AN AUTORADIOGRAPH PREPARED FROM ANEXP'LANTCULTUREDFORFIVEDAYSINMED II comma 0.2 pc./ml. or DL-LEUCINE-Z-C V. 178 179 of exposure was so light that it was impossible to take a photograph of this autoradiograph. This indicates that the secretory explants not only take up materials and accumulate them in areas of active secretion but can also transfer them to the duct system. 5. R _0,. 0m.- -. ' in “page. an: ‘ 7“ or: _.-o‘-. -6 ° Hem-c T’ e c- t ' C Cl .. —o o Laurels Due to the interesting results obtained on the‘Y’-globulins and albumins reported in the work on the injection of labeled compounds into lactating rabbits. it was decided to try to demonstrate incorporation of radioactive'T’-globulin and albumin into secretory mammary tissue ex- plants in_zitrg. Medium.II was prepared and Clu-V’-globulins and Clu— albumin were added to portions of it to give an activity of 0.1.pc./ml. of medium. Explants from the same lactating animal were cultured in these mediums. Also, explants from this animal were placed in sterile water at 50°C for one minute and then cultured in the same mediums. This heat treatment of explants was also carried out on explants cultured in the radioactive leucine and glutamic acid mediums. Figure 70 is a photomicrograph of a autoradiograph prepared from an explant cultured in the medium containing Clu-V’-globulin (0.1 uc./ml. of medium). This photomicrograph shows quite clearly that the activity of the Clu-W’-globulin is concentrated in the secretory alveolar cells. This experiment was duplicated on explants from another lactating guinea pig. This has been done on a total of 16 explants from two animals. The explants cultured in the medium containing C1“ albumin.(0.l.pc./ml. of medium) did not show any exposure of the emulsion, although they were secretory upon histological examination of the explants. Also, the heat treated explants showed no exposure of the emulsion in any of the FIGURE 70 . PHOTOMICROGRAPH OF AN AUTORADIOGRAPH PREPARED FROM AN CULTUREDFORFIVEDAYS INMEDIUMII CONTAINING 0.1 pc./m1. OF 01 LABELED T-GLOBULIN. 180 181 -01”, Ola-\ruglobulin. experiments with leucinewzaclu. glutamic acid-2 or Cluwalbumin. On the basis of these results. it may be concluded that‘Y'-glohulin can be taken up and utilized by actively secreting mammary tissue. Also. that albumin is not taken up to any major extent by these explants 13.11329. B. Discussion 0 a . - 1. ’“9 zfl.u ‘ -' _0,I 0° . -. 00 o 1.. , u- -00 i-.." Mammary Tissue lg Vitgg in Which Negative Results'were Obtained There are many difficulties encountered in tissue culture. Some of these have been described previously in the methods section, such as preparation of glassware. mediums. explants. and bacterial contamination. Over the years. mammary tissue has been one of the most difficult tissues to culture. Lasfargues (1957a) reported the successful maintenance and proliferation of mammary epithelium removed from adult C-57 strain mice prior to the tenth day of pregnancy. The epithelial cells were dispersed by incubation with collagenase for l to 2 hours. The free cells were then cultured in various mediums. He reported that serum from human placenta gave the best results, and noted a proliferation of sheets of epithelial cells. He states that these sheets of epithelial cells were organized into a typical mammary pattern. However. the photomicrographs in his paper do not appear to have a typical organization when compared to a histological preparation of mouse mammary tissue. Elias (1957) reported that organ cultures of mammary tissue taken from CBH/He CR GL strain mice could be maintained by means of synthetic "199" medium enriched with hormones. He further showed that it was possible to stimulate very slight secretion in these explants with his high level medium. He enriched his medium with estrone. progesterone, 182 \1 cortisol. growth hormone. and mammotrOpic hormone in two concentrations. The high concentration contained 0.2, 2.0, 8.0, 140, and luO ug./ml. of medium, respectively. The low concentration contained 0.02, 2.0, 2.0, 20. and 20 ug./ml. of medium. respectively. These mediums of Elias (1957) provided the basis for the mediums ultimately developed for the guinea pig (Tables 21 and 22). An attempt was made to duplicate the work of Elias (1957) on mice. In this study. C—57 strain mice, albino rats, and New Zeeland white rabbits during mid-pregnancy or a few days before parturition, and during various stages of lactation were used. The mediums and methods of cul- ture were identical with those of Elias (1957). This work was completely unsuccessful. There are a number of possible explanations for the failure of these experiments. It is to a certain degree surprising that Elias (1957) has been able to initiate lactation in_yitzg in the presence of estrogen and progesterone. Nelson (1936) has postulated that estrogen has a direct inhibitory effect on the lactational performance of the mammary gland. Meites and Turner (l942a,b.c) have postulated that there are stimulatory as well as inhibitory factors involved in the initiation of lactation. They have shown that estrogen in the intact animal can. significantly increase the pituitary prolactin content and that the level of prolactin in the blood of the rabbit is increased. It is possible that estrogen at some doses has an inhibitory effect on milk secretion as postulated by Nelson (1936). There is no doubt about the effect of prolactin on mammary tissue. The results of Elias (1957) might be explained by the fact that the concentration of prolactin was high enough to overcome any inhibi- tory effects of estrogen in the medium. This is compatible with the theories of Meites and Turner (l9u2a,b.c) and with the results of 183 Sgouris and Meites (l95h, 1955) on the rabbit. The failure to obtain positive results with the mediums of Elias (1957) on mice, rats, and rabbits might be explained by the fact that the concentrations of estrogen and progesterone were too high to be overcome by the level of prolactin added to the mediums. The prolactin used by Elias (1957) was supplied by Dr. C. H. Li of the University of California, Berkeley (Li preparation L2738E). It is possible that Li's preparation is much purer than Armor's Fanlitar, used in this work, and thus contained more prolactin per unit weight of material. There are other factors which might have contributed to the failure of these initial trials. It is possible that there are species dif- ferences involved. It is also possible that there are slightly different requirements of the absolute levels of hormones necessary to maintain various types of mammary tissues and also the ratios of these hormones might vary slightly. ImprOper preparation of glassware might have caused a toxicity which was not apparent to the investigator during this early work. Also, this work was the first attempt in the area of tissue culture. It seems reasonable to believe that inexperience might also have contributed to the lack of positive results in this carry work. 2. IA: ; oomzo 0 U; Non-Secretory Gui a: v; , ; ; ‘ . ._ nea Pig Mammag Tissue lantim‘ The early difficulties encountered in tissue culture experiments prompted trials with various combinations, levels, and ratios of hormones.' Lyons gtflgl. (1958) have shown conclusively that prolactin and corticoids are essential for the initiation of lactation in the fully developed mammary glands of hypophysectomized, oophorectomized, 184 adrenalectomized LongeEvans rats. Elias (1957) also reported that he was able to maintain his cultures with prolactin and cortisol alone. Elias (1959) and Trowell (19 59) reported that insulin had a beneficial effect on mammary tissue eXplants and that it alone was capable of mainn taining the tissue to some extent. Trowell (1959) also reported that a high oxygen gas phase was beneficial in the maintenance of rat mammary explants. Lasfargues (l957a.b) described the proliferation of sheets of mammary epithelium grown in various types of serum. On the basis of these reports of mammary tissues grown in a variety cf media and on the basis of Lyons9 at al. (1958) results, it was decided to prepare the medium described in Table 21. This medium was first used to test the maintenance of rabbit mammary tissue in,ziizg. There was very minimal maintenance of these explants. The alveoli were degenerate but there were very normal viable epithelial cells in the explants. In some respects this was discouraging but not completely so. A decision was made to attempt the cultivation of guinea pig mammary tissue. This was done first with mammary tissue from a non-lactating guinea pig. Excellent maintenance of this tissue was achieved. Various other modifications of this medium were then tried to improve it with the ultimate aim of initiation of lactation in,x11:g. The level of hydrocortisone was doubled. This caused harmful effects in that the epithelial cells were maintained but the alveolar organiza- tion was destroyed. This finding that high levels of hydrocortisone can be harmful has been confirmed by Larson (1959) in his work with the cultivation of bovine mammary epithelium. Higher levels of prolactin and insulin did not appear to be of additional benefit for the main- tenance of non-secretory guinea pig mammary tissue. Medium I without 185 rabbit serum will maintain nonusecretory guinea pig mammary tissue. However, the explants do not look as good as those cultured in the presence of the rabbit serum. Growth hormone did not appear to have any beneficial effects on the maintenance of these non-secretory ex- plants. This work has provided a medium for good maintenance of non- secretory guinea pig mammary tissue but it has not resulted in the initiation of lactation.in‘yitrg. Next, mammary tissue from a lactating guinea pig was cultured in medium I. It was surprising to find that the explants from this tissue were completely degenerate. This indicated that the requirements for secretory'mammary tissue were different than the requirements for non- secretory mammary tissue. From a biochemical point of view, it might be expected that the nutritional requirements would be different. The levels of hydrocortisone, pzolactin, and insulin were doubled over the levels reported in Table 21 to prepare medium II described in Table 22. EXplants prepared from secretory guinea pig mammary tissues were cultured in medium II. At the end of the culture period, it was found upon histological examination that the tissue was highly secretory. The secretion was as good or better than the secretion found in the nonmcultured control. Again, variations of this medium were prepared and evaluated. None of these variations maintained the secretory tissue any better than the medium reported in Table 22. Explants were prepared from non-secretory guinea pig mammary tissue and cultured in medium.II. The aim was to initiate lactation in this non-secretory mammary tissue in.x1$zg. The explants from this none secretory mammary tissue were well maintained, but there was no initi- ation of secretion in these eXplants. 186 The serum used in these media was drawn from healthy male rabbits. It is doubtful that the endogenous hormones of this serum had any bene- ficial effect on the cultures. The serum was added in the same con- centration (10%) to the control medium as well as mediums I and II. All tissues cultured in the control medium (without hormonal additions) were completely degenerate upon histological examination. These results on the development of adequate mediums for the main- tenance of non-secretory and secretory guinea pig mammary tissue inbyitzg show conclusively that the hormonal requirements of secretory and non- secretory guinea pig mammary tissues inlxitrg are different. The requirements for prolactin, hydrocortisone, and insulin are two times as high for the maintenance of secretory tissue as they are for the maintenance of non—secretory tissue. Unfortunately, there are no other reports on the cultivation and maintenance of secretory mammary tissue by the organ culture technique in the guinea pig. Thus, no direct comparisons can be made with the work of others. The hormonal requirements of guinea pig mammary tissue in,yitzg are in agreement with the hormonal requirements reported for bovine mammary epithelium by Larson (1959), Ebner and Larson (1958, 1959), and Hoover sl.fil. (1959). The hormonal requirements are also in agree- ment with those reported by Elias (1957, 1959) and Elias and Rivera (1959) for mouse mammary. The absolute amounts of the hormones required are difficult to compare in the case of prolactin since the activities of Li's preparations used by Elias (1957) are not reported. However, the quantity of insulin used by Elias and the quantity in medium I (Table 21) are the same. Also, Elias (1957) used 8 pg./ml. of cortisol and medium.I contained the-same amount. . 187 Although a medium.was not developed which would initiate secretion in a non-secretory mammary tissue, the results are encouraging. Table 25 presents data which show that the secretory activity of some of the explants could be enhanced by medium II. Thus, there is some evidence for a hormonal stimulus increasing the lactational performance of guinea pig mammary'tissue explants in filing. On the basis of these data, it is felt that the initiation of lactation for the purpose of evaluating hormonal requirements in_xitrg in guinea pig mammary tissue can be accomplished. It is believed that the right combination or ratio of prolactin, hydrocortisone, and insulin would be effective. This is a very extensive task when all of the possible combinations are considered. It is interesting to draw some general comparisons between the levels of prolactin used in medium II and levels of prolactin that will produce a response in_xiyg. Meites et a1. (1941) demonstrated a re- sponse in the crOp sacs of 50% of pigeons injected intradermally over the crop sacs with a total of 0.00624 I.U. of prolactin. Medium II contained “.8 I.U. of prolactin per ml. This is a much higher level than Meites (lth) has shown necessary for pigeon crop proliferation. Meites and Turner (1950) have shown that the pituitaries of various mammals contain from 1.11 to 29.5 I.U. of prolactin per mg. of pituitary tissue. 0n the basis of these data, it appears that mammary tissue in 21122 requires much more prolactin than mammary tissue inhxixg. It should be pointed out that the explant is dependent primarily upon diffusion of substances from the medium into the tissue. This might in part eXplain the apparently large difference between the ingyiyg_and ‘in,yitzg requirements. 188 The photomicrographs demonstrate clearly the hormonal dependence of guinea pig mammary tissue in_xitrgo They also demonstrate the differences in the hormonal requirements of secretory and non—secretory mammary tissue. On the basis of the work with secretory mammary tissue, it appears that the level of secretion within mammary tissue is not as important as whether it is secretory or nonosecretory. As long as there is some secretion, it appears that it is possible to increase the rate of secretion inbyitzg by means of prolactin. hydrocortisone. and insulin. The finding that guinea pig mammary tissue can be maintained in a state of active secretion is important. It provides a system for long term biochemical studies on the lactating mammary gland isolated from the influences of the rest of the body. The need for metabolically active tissues isolated from the hormonal influences of the organism and from other tissues and the organism as a whole has been discussed by Fell (195u)° The results of these in_yit;2 experiments demonstrate quite clearly the importance of organ cultures in the study of endocrinology and bio- chemistry of the mammary gland. These studies with isotOpes on organ cultures of the mammary gland are very interesting. They have provided some insight into the way in which the mammary gland obtains materials for synthesis of milk constituents. This is the first work with tissue culture which has demonstrated the ability of mammary epithelium to take up and utilize native proteins (Y'-globulins)o Heating the explants to 50° centigrade appears to destroy their ability to take up‘Y’-globulins. leucine, and glutamic acid. It seems probable that there are enzyme systems involved in the uptake of these substances by the mammary gland. 189 It appears as though there is a different mechanism or process within mammary tissue for the uptake of'T’-globulins and albumins and their ultimate appearance in milk. This in 11329 work described above lends support to the discussion of the mechanisms involved in the trans- port of Y-globulins and albumins from the blood to the milk at the end of the section on the results of the injection of radioactive materials into lactating rabbits. Studies on the organ culture of mammary tissue are relatively new. As demonstrated by this work, the organ culture of mammary tissue can extend our knowledge of the endocrinology, biochemistry, and physiology of the mammary gland. SUMMARY 1. The objectives of this research were: a. To provide quantitative data on the precursors of caseins, B —l.actoglobulin and O(-lactoglobulin of rabbit milk. b. To provide quantitative data on the relationship between lF-globulins of blood and immune globulins of rabbit milk. c. To provide quantitative data on the relationship between albumin of blood and the "blood" albumin of rabbit milk. d. To deve10p a synthetic medium enriched with hormones which would be capable of maintaining non-secretory mammary tissue in.yi&rg. e. To develop a medium enriched with hormones capable of initiation of lactation in non-secretory mammary tissue inbxitzg. f. To develop a medium enriched with hormones capable of,maintaining secretion in secretory mammary tissue in,yitzg g. To study the ability of secretory mammary tissue to take up amino acids and blood proteins inbxitzg. 2. The methods utilized in this research involved the blood fraction- ation by the procedures of Cohn et,al,'{l950). ,Albnmins,‘7’-globulins and crude fractions of O(-globulins and fifglobulins were obtained by this method. Paper electrophoresis patterns were run on these fractions and on whole serum to prove their identity and establish their homo- geneityol Two male New’Zeeland white rabbits were injected with 018 labeled BaC03. The animals were placed in a chamber designed and built to trap expired C1“ labeled C02. Six hours post injection, the animals were bled and cl“ labeled serum protein fractions were isolated by the method or Cohn it 2.10 (1959). 191 Classical ammonium sulfate fractionation procedures were employed for the fractionation of the B -lactoglqbulin, OQ-lactalbumin and "blood" albumin in rabbit milk. Rabbit milk caseins were precipitated by ad- justing the pH to their isoelectric point which.was found to be #.3. It was found that the immune globulin fraction of rabbit milk could be isolated by readjustment of the acid whey to pH 6.0. A method for paper electrOphoresis of rabbit whey and rabbit milk proteins was developed which gave very good results. This method was the precoating of the paper strips with gelatin9 thus preventing the absorption of the whey proteins into the paper strips. The percent of leucine and glutamic acid were determined in the blood protein and milk protein fractions. This was accomplished by hydrolysis of the proteins and by column chromatography. The eluents from the column were subjected to paper chromatography as a purity check. Aliquots of eluents were treated with ninhydrin and the amount of amino acid determined colorimetrically. Free blood leucine and glutamic acid were isolated from the blood by column chromatography. The chemical purity of DL-leucine-Z—Clu and Dlpglutamic acid-Z-Clu was established by paper chromatography and subsequent counting of the paper strips in a gas flow strip counter. A method was developed for the milking of rabbits. This was accomplished by injecting 1.0 I.U. of oxytocin into the marginal ear vein. A beveled glass tube was placed over the nipple immediately following the injection. The tube was connected to a reservoir for collection of milk which was in turn connected to a water aspirator. A negative pressure of 15 mm. of Hg. was maintained in the system. Vigorous massage of the mammary gland from the periphery toward the 192 base of the nipple was applied together with periodic interruption of the suction by partial removal of the tube from the nipple. Blood samples were obtained from the rabbits by heart puncture into the left ventricle. Dleleucineu-Z-Clu', DL-glutamic acid-Z-Cll" and (flu-protein fractions were dissolved in a minimal amount of phosphate buffer (pH 7.2) and injected by marginal ear vein into 10 lactating rabbits. Blood and milk samples were obtained at 2, 6, 12, 24, 36, #8 and 72 hours post injection. The blood and milk samples were fractionated into the various serum and milk protein fractions described above. The blood and milk protein fractions were dialyzed against dis- tilled water until free of reagents. The fractions were then 1y0phillized. Glutamic acid was isolated from the protein fractions by acid hydrolysis and precipitation of the chloride of glutamic acid. The protein, free glutamic acid and leucine samples were counted by Ig-liquid scintillation counting procedures. Mammary tissue was cultured by the organ culture method. The explants were placed on treated rafts of cellulose-acetate. The rafts were floated in the synthetic medium.in a watch glass in a Petri dish. The explants were cultured for 5 days. The medium was changed when the pH drOpped below 7.0. Histological sections of the explants were prepared and stained with iron hemotoxylin and eosin in the usual manner. Media for mammary tissue culture were prepared from Parker's ”199” synthetic medium. Prolactin, hydrocortisone and insulin‘were added at 2 concentration levels. The levels of these hormones for mammary tissue maintenance were lho, 8 and 70 ug./m1. of medium, respectively. The levels of these hormones for maintenance of secretion in mammary 193 tissue were 240. 16 and 140 ug./ml. of medium. respectively. The media were gassed with 95% 02 and 5% cog. DL—leucine—Z-Clu', DL-glutamic acid- 2aclL‘. Clu-Yglobulin and cluaalbumin were also added to portions of the media for maintenance of secretion inixitrg. Autoradiographs were prepared from explants cultured in media con- taining C14 labeled amino acids or proteins. The sections of the tissues were mounted on microscope slides and stained with hemotoxylin and eosin in the usual way. The slides were coverslipped‘with celloidin. A thin film of Eastman Kodak emulsion.was applied in the darkroom. The slides were sealed in a light proof container and placed in the refrigerator for exposure. The exposed autoradiographs were developed with dektal. fixed in acid fix and washed with tap water. They were then dehydrated and coverslipped in the usual manner. 3. The major results and findings of this work are as follows: lfllkhfinisin_§indiea a. In Experiment 1 in which 1+5 no. of DL-leucine-Z-Clu was in- jected into a lactating rabbit, the free blood leucine decreased at an extremely rapid rate during the first 10 minutes post injection. The calculated value at zero time was 157.2 pc./mm. in the extracellular water. At 10 minutes, the experimentally determined value was 3.7 "pc./ mm. The equation for this disappearance of free blood leucine is A = Ace'00375t. The t% value is 1.85 min. and the rate constant is -0.375 min.'1. After 10 minutes the rate constant changes very drastically to a value of -0.00169 him-1 with a 12% of n09.5 min. A system to explain these interesting findings has been postulated. On the basis of these data and the shape of the curve, it has been postulated that the leucine injected has been sequestered somewhere 19h in the animal's body. likely possibilities are the reticulo-endothelial system. the intracellular pool and plasma protein binding. After two hours the leucine from this sequestered pool appears to return to the extracellular leucine pool at a rate slightly less than the rate of removal of free leucine from the extracellular pool. Thus, it appears that only a small proportion of the injected DL-leucine-Z-Clu'was avail- able for synthesis into milk proteins at any particular time. Thus, an attempt was made to establish the parameters for leucine as a direct precursor of milk proteins. It appears that the curve for free blood leucine as determined in Experiment 1 represents the amount of free blood leucine available for incorporation into milk proteins. b. The data in Experiment 1 show that the leucine incorporated into caseins. p ~lactoglobulin and o(-lactalbumin of rabbit milk are derived from the free blood leucine. The curves presented fit the criteria for precursor product relationships very well. Further, it takes an average of h hours for a free blood leucine molecule to be incorporated into a milk protein and transferred to the ducts ready for excretion as a milk constituent. c. The ratios of the specific activities of blood Y’-globulins and milk immune globulins in Experiment 1 show that at least 73. 5% of the milk immune globulinS'were derived directly from the blood nglobulins. ' The 'immune globtflins of. the milk were not synthesized within the mammary gland since the specific activities of leucine incorporated into the immune globulins of milk and the specific activi- ties of caseins, fiB-lactoglobulins and.0<-lactalhumins are different. d. It is also concluded from Experiment 1 that at least 77.h% of the ”blood” serum albumin in rabbit milk is derived directly from 195 the albumin of blood plasma. This is based on the ratios of the specific activity in these 2 protein fractions. The blood T-globulins and albumin rapidly achieve equilibrium with the milk immune globulins and "blood" serum albumin of milk. The ratio of the specific activity of the immune globulin from milk to T—globulin from blood at 2 hours post injection was 0.916 and the ratio of specific activity of "blood” serum albumin from milk to albumin from blood at 2 hours post injection was 0.900, thus demonstrating the rapid equilibrium between these blood proteins and their corresponding milk proteins. e. Other experiments with DL-leucine-Z-C1le and DL-glutamic acid.- 2-01” completely substantiate the results on the first experiment with DL—leucine-Z-Clu. It is noteworthy that all of the glutamic acid incor- porated into caseins, fl -lactoglobulin andN—lactalbumin of the milk of lactating rabbits apparently comes directly from the free blood glutamic acid. f. Experiments were designed to evaluate the importance of plasma proteins as precursors of milk proteins by the injection ‘of C1]+ labeled -plasma protein fractions into lactating rabbits. When C1“ labeled Y-globulins were injected into 2 lactating rabbits, a close correlation was obtained between the specific activities of the Y-globulins and milk immune globulins. The time required to establish equilibrium be- tween the blood and the milk fractions was slightly more than 2 hours in one animal and slightly more than 6 hours in the other animal. After equilibrium had been reached, it was calculated from the ratios of the Specific activities of milk immune globulins toY-globulins that 93.14% of the milk ilmnune globulin in the milk of one animal and 98.2% of milk immune globulin in the milk of the other animal were derived from the Y—globulin in the plasmas. 196 g. 'When cl“ labeled albumins were injected into 2 lactating rabbits, the relationship between the plasma albumins and "blood" serum albumin in milk was found to be similar to that for'T'-globu1ins and milk immune globulins. In this case, it took between 6 and 12 hours to establish an equilibrium between the blood plasma albumin and the "blood" serum albumin from milk. This was true in both animals. After equilibrium had been established, it was calculated from the ratios of the specific activities of "blood" serum in milk to the specific activities of albumin in plasma that 92.2% of the "blood" serum altumin in the milk of one animal and 98.2% of the ”blood" serum albumin in the milk of the other animal were derived from the albumin in the plasma. h. Studies on the injection of C14 labeled 0(- and B -globulins from blood plasma indicated that they'were of little importance as pre» cursors of milk proteins. Also, no milk protein fraction was detected which was derived directly from.these two blood protein fractions. lg Vjtzp Studies with Mammary Tissue Cultures a. It was shown that'Y'-globulin can be taken up by mammary tissue in active secretion and concentrated in the alveolar cells in.xitzg. This ability is destroyed by heating the explant to 50°C just prior to cultivation. b. Prolactin (ZhO ug./ml.), hydrocortisone (l6 ug./ml.) and .insulin (luo ug./ml.) added to a synthetic medium are capable of main- taining active secretion in mammary.tissue in,yitrg. This was shown by histological preparations and by the active uptake of leucine and glutamic acid and their apparent incorporation into mammary secretory products. c. Albumin is not taken up by the secretory mammary tissue inkzitzg. 197 d. Heating explants to 50°C for 1 minute destroys the ability of mammary'tissues to take up'7'wglobulins, leucine and glutamic acid e. Prolactin (lMO ug./ml.), hydrocortisone (8 ug./ml.) and inm sulin (70 ug./m1.) in a synthetic medium are capable of maintaining nonwsecretory mammary tissue inbyitrg. Seventyathree percent of the total lobuloealveolar tissue was maintained in 108 explants prepared from 8 nonwlactating guinea pigs. f. Prolactin (240 ug./m1.), hydrocortisone (16 ug./m1.) and insulin (70 ug./m1.) in a synthetic medium are capable of maintaining secretory mammary tissue ig_vitrg. Seventy percent of the total lobuloe alveolar tissue was maintained, and 55.b% of the total lobulomalveolar system was maintained in an active secretory state in 180 explants prem pared from 12 lactating guinea pigs. . g. The data obtained on milk immune globulins and "blood" serum albumin in milk and the data obtained by the addition of Cl“ labeled 7’-globulins and albumins to tissue culture media indicate that there is a different mechanism by which the mammary epithelium incorporates these proteins into milk. It is postulated that the mammary'gland actively takes up'1'-globulins or is permeable to'7'-globulins, and that it does not actively take up plasma albumin or that the mammary gland is relatively impermeable to altmmin. BIBLIOGRAPHY Arden. I. A.. and Tarver, H. Plasma Protein I. Loss from Circulation and Catabolism to Carbon Dioxide. J. B121. ghgm. 1902769_780, iVEla. Abiwu. I. A.. and Tarver, H° lasma Protein II. Relationship Between Circulating and Tissue Protein. J. 2121. Chem. 190278lm790, 1951b. Askonas. B. A., Campbell, P. N., Godin, C., and Wbrk, T. S. The Bio- synthesis of Proteins. 3. Precursors in the Synthesis of Casein and flelac'toglcbulin. mm. 1. 61:105-115. 1955. Askonas, B. A.. Campbell, P. N.. Humphrey, J. H. and Work. T. S. The Source of Antibody Globulin in Rabbit Milk and Goat Colostrum. Blame lo 563.597.6019 1954. Askonas, B. A., Campbell, P. N.. and werk, T. S. The Biosynthesis of Proteins. 2. Synthesis of Milk Proteins by the Goat. Bigghgm. i. .58 g 326“‘331~ 9 19.540 Askonas, B. A., and Humphrey, J. H. Formation of Specific Antibodies and‘Y'eGlobulin ln_Ei;1g. Bigghgm.‘£. 68:252w261, 1958. Aten, A. H. W. Jr., and Hevesy, G. Formation of Milk. Natu:e. 1&2: 111-112, 1938. Asimov, G. I. Some Processes Accompanying the Secretion of Milk. Int. 2.2.2.11 9.11.11. 1119.. 1:15-19. 1959. Barry, J. M. The Source of Lysine, Tyrosine, and Phosphorus for Casein Synthesis. ,1. 2°01. Chem. 1952795m803, 1952. Barry, J. M. The Use of Glutamine and Glutamic Acid by the Mammary land for Casein Synthesis. Bigghgm.,l. 638669-676, 1956. Barry, J. M. The Source of Proline for Casein Biosynthesis. Bigghem. i. 703177~179a 195 . Barry, J. M. The Precursors in the Blood Stream of the Proteins of Milk- 2.191. 29X. .821. 2. 1A92380~391. 1958. Beyer, K. H., wright, L. D., Russo, H. F., Skeggs, H. R., and Patch, E. A. The Renal Clearance of Essential Amino Acids: Tryptophane, Leucine, Isoleucine, and Valine. Am. J. Physiol. 146:330-335, l9h6. Black, A. L., and Kleiber, M. Level of Fixed Carbon in Amino Acids of Casein Measured in the Intact Dairy Cow. i. 2121. Qhem. 2102895- 9029 1954. Blackwood, J. H. The Absorption of Milk Precursors by the Mammary Gland. III. The Relation of Amino-Acid Absorption to Protein Synw thesis. Bigghem. J. 26:77za777, 1932. 199 Block. R. J.. and'weiss. K. W. "Amino Acid Handbook." Charles C. Thomas. Publisher. Springfield. Illinois. pp. 76~779 1956a. Block. R. J.. and weiss. K. W. "Amino Acid Handbook." Charles C. Thomas. Publisher. Springfield. Illinois. U.S.A. pp. 296-297. l956b. Bouckaert. J. H.. Oyaert.‘w.. Peeters. G.. and Sierens. G. Uptake of Plasma Amino Acids by the Perfused Isolated Cow’s Udder. Axghp int. Bananas. 93 3W3 W49 9 1953 . Campbell. P. N.. and Work. T. S. The Biosynthesis of Protein. 1. Uptake of Glycine. Serine. Valine and Lysine by the Mammary Gland of the Rabbit. Bia1hsm. J. 52:217w227. 1952. Cary. C. A. AminowAcids of the Blood as the Precursors of Milk Proteins. 13.0 Mo Qbfimo 143247714899 197400 Chen. J. M. The Cultivation in Fluid Medium of Organized Liver. Pancreas and Other Tissue of Fetal Rats. Exp. Qfill.E§§. 72518~529, 195“. Cohen. 8.. Holloway. R. 0.. Matthews. C.& and McFarlane. A. S. Distrim bution and Elimination of *1- and 1 C—Labeled Plasma Proteins in the Rabbit. s: A... chem. g. 6231.13-15». 1956. Cohn. E. J.. Curd. F. R. N.. Surgenor. D. M.. Barnes. B. A.. Brown. R. K.. Derouaux. G.. Gillespie. J. M.. Kahnt. F. W.. Lever. W. F.. Iiu. C. H.. Mittelman. D.. Mbuton. R. F.. Schmid. K.. and Uroma. E. System for the Separation of the Components of Human Blood: Quantitative Pro~ cedures for the Separation of the Protein Components of Human Plasma. J. Am. Chem. Elsie 723%5--4?Lb 1950. Colas. J. LeBars, H. Simonnet. H.. and Sternberg. J. Recherches sur la biochimie des composes phosphoes du lait III.. Les protéines phos~ phorées de la glands mammaire en lactation. Compt. rend. 232:11h8— 1151 . 1950. Coulson, E. J.. and Stevens. H. The Serological Relationship of Bovine Whey Albumin to Serum Albumin. J. Bil-21° Shem. 1872355-363, 1950. Crowther, C., and Raistrick. H. A Comparative Study of the Proteins of the Colostrum and Milk of the Cow and Their Relation to the Serum PrOteins 0 We in 10 3 “Bu-1+? 9 1916 9 Dixon. F. J.,‘Maurer. P. H., and Deichmiller, M. P. Half-Lives of Homologue Serum.Altmmins in Several Species. E:gg..§gg..£xp§lm 2111. and Med. 83:287-288. (1953). Dixon, F. J.. Talmage, D. W., Mauer. P. H.. and Deichmiller. M. The Half-Life of Homologue Gamma Globulin (Antibody) in Several Species. lo E23111. M11. 963313-318. 1952. 200 Dovey, A., Holloway, R. C., Piha, R. 3., Humphrey, J. H., and MbFarlane, A. 8. Protein Turnover in Rabbits. In "Proceedings of the 2nd Radio- isotope Conference." Oxford. Butterworths, Tendon. 3379 1954. Ebner, K. E., and Larson, B. L. In 113:9. Cultivation of Bovine Mammary Gland in Tissue Culture. .1. 12am égl. “1272791959, Ebner, K. E., and Larson, B. L. Maintenance, Proliferation, and Bio- synthesis of Milk Constituents by Mammary Cells in Tissue Culture. 51° m .5310 “23916r9179 19590 Ehrlich, P. Beitrage zur Kenutuis der Milch immunisierter Tiers. 2. H33. Infelstkr. 12:183~l98, 1892. Elias, J. J. Cultivation of Adult Mouse Mammary Gland in Hormone- Enriched Synthetic Medium. §212nga. 126:8Q2-8h4, 1957. Elias, J. J. Effect of Insulin and Cortisol on Organ Cultures of Adult Mouse Mammary Gland. Ezgg.'§ggp Empfinp Biol. and,flgd. lOl:500-502, 1959. Elias, J. J., and Rivera, E. Comparison of the Responses of Normal, Precancerous, and NeOplastic Mouse Mammary Tissues to Hormones In limo gangs: Rea. 192505-611. 1959. Espe, D. L., and Smith, V. R. "Secretion of Milk," 4th Ed. The Iowa State College Press, Ames, Iowa. p. 99, 1952. Fell, H. B. The Effect of Hormones and Vitamin A on Organ Cultures. Am» Hm Kerk Aged. s21. 58:1183-1187. 1954. Fell, H. B., and Robison, R. The Growth, Development and Phosphate Activity of Embryonic Avian Femora and limb Buds Cultivated Inbiiizgo W0 .110 2337674781“, 19290 Fink, R. M., Enns, T., Kimball, C. P., Silberstein, H. E., Bale,'W. F., Madden, S., and Whipple, G. H. Plasma Protein Metabolismp—Normal and Associated with Shock. Observations Using Protein Labeled by Heavy Nitrogen in Lysine. 1. m. Mad. 80:455—1475, 19%. Fischer, E. Ueber die Hydrolyse des Caseins durch Salzsaure. ‘L. magi. chem. 33:151-176. 1901. Fee, C. Sull'origine del Lattosia, della Caseina e del Grosso del Latte. Arab. E15191. 102uoz-u2u, 1912. FOlley, So Jo Iac‘tationo 3191‘ m0 15:14’21-‘463, 19%0 Folley, s. J. Biochemical Aspectsof Mammary Gland motion. 3191. 32m. mom-351+. 1949. _- 201 Folley, S. J., "The Physiology and Biochemistry of Lactation." Charles C. Thomas, Publisher, Springfield, Illinois, U.S.A.9 pp. 130-137, 1956. Folley, S. J., and Greenbaum, A. L. Changes in the Arginase and Alka~ line Phosphatase Contents of the Mammary Gland and Liver of the Rat During Pregnancy, Lactation and Mammary Involution. Bigchgm. g. bl:26lu269, 1947. Friedberg, F., Tarver, H., and Greenfierg, D. The Ldstzitmtioanattern of Sulfuerabeled Methionine in the Protein and the Free Amino Acid Fraction of Tissues after Intravenous Administration. J. Biol. Chem. 1733355~36l, 1948. Germuth, F., Oyama, J., and Ottinger, B. The Mechanism of Action of l7~Hydroxyull~Dehydrocorticosterone (Compound E) and of the Adreno- corticotropic Hormone in Experimental Hypersensisitivity in Rabbits. £0 We Mo 91"“3139‘1709 1951. Graham, W. R., Jr. The Utilization of Lactic Acid by the Lactating Mammary Gland. .1. £1521. £11233. 12221-99, 1937. Graham,'w. R., Jr., Houchin, O. B., Peterson, V. B., and Turner, C. W. The Efficiency of the Mammary Gland in the Production of Milk. Al”: J.. was. 122:150—-153. 1938. Graham, W. R., Jr., Kay, H. D., and McIntosh, R. A. A Convenient Method for Obtaining Bovine Arterial Blood. P299. 3215 §Q§. (Lendgn) E. 1203319629. 1936. Graham, W. R., Jr., Peterson, V. E., Houchin, O. B., and Turner, C. w. The Utilization of Fractions of the Nitrogen Partition of the Blood by the Active Mammary Gland. .1. moi. Eben. 1222275-283, 1938. Gross, J., Bogoroch, R., Nadler, N. J., and Leblond, C. P. The Theory and Methods of the Radioautographic Localization of Radioelements in Tissues. Am. in We Radium Ingram. 65=l+20-458 (1951)- Hammond, J., and Marshall, F.H.A. "Reproduction in the Rabbit." Oliver and Boyd. Edinburgh, pp. 122-125, 1925. Hansen, R. G., and Phillips, P. H. Studies on Proteins from Bovine Colostrum I. ElectrOphoretic Studies on the Blood Serum Proteins of ColostrumpFree Calves and of Calves Fed Colostrum at various Ages. J. 5191. Qhfimp 171:223-227, 19h7. Hansen, R. G., Potter, R. L., and Phillips, H. Studies on Proteins from Bovine Colostrum II. Some Amino Acid Analyses of a Purified Colostrum Pseudoglobulin. lo ELQlo Qhflrh 1713229-232, 1947. Hardy, M. H. The DevelOpment.ln|Vitzg of the Mammary Glands of the Mouse. l, Anai, 843388-393: 1950- 202 Henriques, 00 B., Henriques, S. B., and Neuberger, A. Quantitative Aspects of Glycine Metabolism in the Rabbit. Eigghfim. l. 60:#O9— “'2‘”? 195.50 Hoover, C. R., Hageman, E. C., Ebner, K. E., and Larson, B. L. Bio- chemical Characterization of Bovine Mammary Cells Grown in Tissue C111.tur‘eo £0 Déi I2 SQio “'2 39179 19590 Howe, P. E. The Use of Sodium Sulfate as the Globulin Precipitant in the Determination of Proteins in Blood. is 2121» Eben. 49:93ulO7, 1921. Hughes,‘w. L. Interstitial Proteins: The Proteins of Blood Plasma and lymph, In “In: Proteins." (edited by Neurath, H., and Bailey, K.), Academic Press, Inc., New Yerk, Vol. 2:663-759, 1954. Humphrey, J. H., and. Sulitzeanu, B. D. The Use of (01”) Amino Acids to Study Sites and Rates of Antibody Synthesis in Living Hyper~ immune Rabbits. Bigghem. i. 68:146mlél, 1958. Jameson, E., AlvarezmTostado, C., and Sortor, H. H. Electrophoresis Studies on Newborn Calf Serum. EZQQ0,§QQQ Expo 212;. and Med. 51:163~165, l9UZ. Jenness, R., Larson, B. L., MCMeekin, T. L., Swanson, A. M., Whitnah, C. H., and Whitney, R. Nomenclature of the Proteins of Bovine Milk. 1. Daizz.§aio 39:536w541. 1956. Kleiber, M., Smith, A. H., and Black, A. L. Carbonate as Precursor of Milk Constituents in the Intact Dairy Cow. ,lo.Einp Qhfimp 1953 707"'712+ 9 1952 o Larson, B. L. Transfer of Specific Blood Serum Proteins to Lacteal Secretions near Parturition. J, Dairy Sci. h1:1033-104#, 1958. Larson, B. L. Personal Communication, 1959. Larson, B. L., and Gillespie, D. C., Origin of the Major Specific Pro- teins in Milk. J, Eigl. ghfim. 2273567-573: 1957. Larson, B. L., and Jenness, R. fl -Lactoglobul_in. 2125211213. W. John'Wiley and Sons, Inc. NeW'York. Vol. 4323-29, 1955. Larson, B. L., and Kendall, K. A. Changes in Specific Blood Serum Pro- tein Levels Associated with Parturition in the Bovine. J. Daizz‘égi. 403659-668, 19570 Lasfargues, E.'Y. Cultivation and Behavior In,Iitrg. of the Normal Mammary Epithelium of the Adult Mouse. ABEL» Egg. 127:117—129, 1957a. 203 Lasfargues, E. Y. Cultivation and Behavior In Vitro of the Normal Mammary Epithelium of the Adult Mouse. ‘Ezoor. Coll Boo. 13:553~ 562 9 195%0 Lauryssens, M., Peeters, G., Coussens, R., and DeLoose, R. Metabolisfins Du Propionatewlelh Dans La Glands Mammaire Bovine Isolée. Argh. Int. pharmacodyn. 1093203~210, 1957. Ieviton, A. Quantitative Ionophoretic Determination of Some Whey Pro~ teins in Skim.Milk. Agzie Eood_§home 5:532e538, 1957. Iintzel, W. Untersuchunsen'fiber den Chemismus der Milchfettbildung in Abhangigkeit von der Futterung. Z. Zucht. Reihbe B.Z. Tierzucht. Zfihktungsbiol. 29:219e242, 193A. Lyons, W. R., Li, C. H., and Johnson, R. E. The Hormonal Control of Mammary Growth and lactation. "Recent Progress In Hormone Research." Academic Press, Inc., New York and London. 142219-2h8, 1958. McCarth , R. D., wong, N. P., and Parks, O.'W. The Metabolism of Serine- 3~~Cl by the Lactating Sheep Mammary Gland. J. Laizzufioi. h281886~ 1887! 1959 o McNaught, M. L., and Folley, S. J. Unpublished, 1958. MEites, J., Bergman, A. J., and Turner, C.'W. Comparison of Assay Methods Using International Standard Lactogen. Ehdoozinologz. 28:707-709, 19u1. Meites, J., and Sgouris, J. T. Can the Ovarian Hormones Inhibit the Mammary Response to Prolactin? Enooozinologz. 53:17-23, 1953. Meites, J., and Sgouris, J. T. Effects of Altering the Balance Between Prolactin and Ovarian Hormones on Initiation of Lactation in Rabbits. Itdasrinclagl. 55:530~534. 195A. Meites, J., and Turner, C.'W. Studies Concerning the Mechanism Controlling the Initiation of Lactation at Parturition. I. Can Estrogen Suppress the Iactogenic Hormone of the Pituitary. Endoorinology. 303711—718, 191428 c Meites, J., and Turner, C.'W. Studies Concerning the Mechanism Controlp ling the Initiation of Lactation at Parturition. II.'Why Lactation is not Initiated During Pregnancy. Endoazinclazz. 30:719-725, l9h2b. Meites, J., and Turner, C. W. Studies Concerning the Mechanism Controlling the Initiation of Lactation at Parturition. III. Can Estrogen Account for the Precipitous Increase in the Lactogenic Content of the Pituitary Following Parturition. ‘Endoozinologxp 30:726u733, l9h2c. 204 Meites, J., and Turner, C. W. Chap. X. in "Hormone Assay." Emmens, C. W. Academic Press, Inc., New York. 237-260, 1950. Moore, 5., Spackman, D. H., and Stein, W. H. Chromatography of Amino Acids on Sulfonated Polystyrene Resins. An Improved System. Anal. gm. 30311851190, 1958. Morgan, J. F., Morton, H. J., and Parker, R. C. Nutrition of Aninnl Cells in Tissue Culture. I. Initial Studies on a Synthetic Medium. Ema. Sea. East. E191. and. Mad. 73:1-8. 1950. Nelson, W. O. Endocrine Control of the Mammary Gland. EM. Box. 16:488-526, 1936. Nichol, J. C., and Deutsch, H. F., Biophysical Studies of Blood Plasma Proteins. VII. Separation of Y-Globulin from the Sera of Various Animals. .510 Mo mo mo 70:80'D-83, 1914‘8. Nikitin, V. N. New Data on the Biochemistry of lactation. Biokhimiya. Niklas, A., and. Maurer, W. Uber die Neubildung einzelner, getrennter Semm-EiweisnFraktioner nach oraler Gabe von 3.'SS--I.~Methionin an Batten. We 2129:1212. 323389-103. 1952. Peeters, G., Coussens, R., De Loose3 R., and Van Den Henck, A. L'Incorporation De D.L. Cystine 5S Dans Les Proteines Du Iait Par La Glands Mammaire. who inj.. W11. 109:415.-424, 1957. Peeters, G., Govaerts, J., and Sierens, G. Métabolisme Du Glucose Dans Le Pis De Vache Isole Et Perfusg. Arch int. W. 10033674739 1957. Peeters, G., and Massart, L. La Perfusion de la Glands Marmaire Isol’ee. Arch. intern. pharmacodyn. 74:8}1947. Peeters, G., and Massart, L. Respiratory Quotient of the Isolated Bovine Mammary Gland. Nam. 1692627-628, 1952 Petersen, W. E. , Sarwar, M., and Campbell, B. Antitryptic Factor and AntibOdy AbSOI'ptiono lo m we 4137269 1958. Petersen, w. B., Shaw, J. C., and Visscher, M. B. Perfusion of the Ebccised Mammary Gland as a Method of Studying Milk Secretion. .1. 2.2121 $.91. 22:439-440. 1939. Petersen, W. B., Shaw, J. C., and Visscher, M. B. A Technique for Pei-fusing Excised Bovine Mammary Glands. J.. Dairy; Sol. 24:139- 146, 1941. 205 Polis, B. D., Shmukler, H. W., and Custer, J. H. Isolation of a Crystalline Albumin from Milk. J. Biol. Chem. 1872349e354, 1950. Reineke, E. F., Peterson, V. B., Houchin, O. B., and Turner, C. W. Studies on the Blood Precursors of Milk Protein. MQ.‘Agr. Exp.‘§ta. 3.2.5.0 mo 296:) 1939. Reineke, E. P., Stonecipher,‘W. D., and Turner, C. W. The Relation Between the Fat and Carbohydrate Metabolism of Lactation, as Indi~ cated by the Respiratory Quotient of the Mammary Gland. Am. J. trio . 132: 535-541, 1941. Reineke, E. P., Williamson, M. B., and Turner, C. W3 Utilization of Glycoprotein of the Blood Plasma by the Lactating Mammary Gland. J. Biol. Chem. 138383~90, 1941. San Clemente, C. L., Huddleson, I. F., Stahl, W. H., Hutchings, In M., and Hamann, E. E. Studies in Brucellosis. II. Mich. State 9911. J‘Afvo Em)- £1.10 Ia .. 333.0 1829 1943. Sansom, B. F., and Barry, J. M. The Use of Asparagine and Glutamine for the Biosynthesis of Casein and Plasma Proteins. Biochem. J. 68: 487— 493. 1958. Schaffer, B. M. The Culture of Organs from Embryonic Chick on Celw lulosemAcetate Fabric. Exp. 9:11. Baa. 11:244e448, 1956. Shaw, J. C. Lactic Acid, Pyruvic Acid, Amino Acids, Acetone Bodies, Oxygen, Carbon Dioxide, and Hemoglobin in Arterial and Mammary Venous Bloods of Cows Under Various Physiological Conditions. .J..2aizx.§aio 293183~1979 1946. Shaw, J. C., and Petersen, W. E. The Ratio of Arterio—Venous Difference of Certain Substances to Quantities Secreted by the Mammary Gland. Am. .1. 21mm. 123:183, 1938a. Shaw, J. C., and Petersen, W. E. Amino Acids and Other Non-Protein Nitrogen Blood Substances in Relation to Milk Secretion. [2:9g. 59;. E232- mo m Med. 383632—6359 1938b. Shaw, J. C., and Petersen,‘W. E. Blood Volume Changes in the Mammary Glam-'10 Erm- EQQO Em. m9].- and. Mad- 423520-528’ 19390 Shaw, J. C., and Petersen, W. E. The Fat Metabolism of the Mammary Gland. J. Dairy Sci. 23:1045—1056, 1940. Sheldon-Peters, J.C.M., and Barry, J.M. The Uptake of Glutamine and Other Amino Acids from the Blood Stream by the Lactating Mammary Gland. W0 filo 63 3 676-679 , 1956. Smith, E. L. The Immune Proteins of the Cow. .Eed. Pros. 52154. 1946a. 206 Smith, E. L. The Immune Proteins of Bovine Colostrum.and Plasma. .1. 2:91 Sham 1642345858. 1946b. Smith, E. L. Isolation and Properties of Immune Iactoglobulins from BOW-he Whey. lo Biol. Chem. 16.53655—676. 1946c. Smith, E. L., Greene, R. D., and Bartner, E. Amino Acid and Carbo~ hydrate Analysis of Some Immune Proteins. i. E191. thm. 164:359~ 366 , 1946d . Smith, E. L. The Isolation of Properties of the Immune Proteins of Bovine Milk and Colostrum and Their Role in Immunity: A. Review. .1. Dairy Sci.- 313127-138. 1948. Smith, E. L., and Greene, R. D. Further Studies on the Amino Acid Composition of Immune Proteins. 1. £121. Qhfim. 1713355e362, 1947. Solomon, A. K. Equations for Tracer EXperiments. J. Qlin. Iny§§.. 2831297-1307, 1949. Trowell, O. A. The Culture of Mature Organs in a Synthetic Medium. 2590 Qfillo Bfiio 16311-8‘1879 1959c Nasserman, K., and Meyerson, H. S. Exchange of Albumin Between Plasma and Lympho Am. :1. Emmi. 165315-26. 1951. Zilversmit, D. B., Entenman9 C., and Fishler, M. C. On the Calculation of "Turnover Time" and "Turnover Rate” from Experiments Involving the Use of Labeling Agents. .J. Gen. Ehysigl. 26:325m331, 1943. APPENDIX > 8 8 2 ‘ 8 1 2 ‘ ‘ ' J1 8 l l . s 3 M .f o H ‘ o . h * \ 8 X ; V§§§ | 3 8 )8 1 ages 5 I .. ‘ 1 g :33 S 3 x; . x. . . 3 8 ‘ 3 k v _ 7 I ,4 U L. r i- m gr. (:8 6. fi‘ \ l I 1 0* I,“ N )0) ’0 515/ 208 60 FIGURE 1. PHOTOGRAPHS OF PAPER CHR TOGRAMS 0F DIPLEUCINE-Z-Clu AND DLnGLUTAMIC ACID—2-01 WITH RADIOACTIVE RECORDINGS. 209 TABLE 1. THE YIELDS, SPECIFIC ACTIVITIES AND PERCENT OF INJECTED DOSE OF SERUM PROTEIN FRACTIONS ISOLATED FROM MALE a RABBITS (L11 AND X--12) INJECTED mm 1.50 flC. of BaC03~01 . Protein Fraction mg. p0. / mg. 95 Injected Dose of 011+ Rabbit L11 TmGlobulins 1.112 0.00033 0.0144 Albumin 0.270 0 . 0026 0.7L» O(-Globulins 553 0.0029 0.1.1 ‘3 ”Globulins 897 0.0021 0.13 Rabbit L12 Y -;Globulins 2 , 045 0 .0025 0.33 Albumin 6 . 3:12 0 .0040 1. 85 (X wGlobulins 1., 118 0 . 0033 0 . 2 5 fl —Globu1ins 877 0.0051 0.30 TABLE '2 . UTILIZED IN EXPERIMENTS 1. THROUGH 1.0 A SUT-II-IARY OF THE TREATMENTS OF THE LACTATING RABBITS 2'10 Exper. An. ‘Wt. Days Igfiwriajs Injegteg Samples No. No. Kg. PP* Material. ‘pc. me. Milk (m1.) Blood (m1.) 1 1-35 5.0 1u-17 L2 u5.0 11.5 11.6 15.0 2 X~32 5.1 10-17 L 45.0 11.5 15.0 15.0 3 xh30 u.0 1n-17 03 150.0 01.2 16.0 15.0 1 xegu 5.2 14-17 G 150.0 41.2 12.5 15.0 5 X-18 5.0 10-13 'yg' 0.13 too 12.1 15.5 6 1-19 u.9 10-13 yr 2.00 800 11.6 13.3 7 1-17 5.5 10-13 A5 5.16 2000 17.5 14.5 8 1-20 4.8 10 13 A 13.20 3000 15.0 13.5 9 1-24 5.2 10-11 cx16 1.63 492 15.0 15.0 10 1-27 3.7 10-11 13'7 2.37 398 15.0 15.0 1 PP = Postpartum 2L = DL-Ieuci.ne~2«--Clu' 30 = DIMGlutamic Acid-chlu LS’ = Cl“ 1’ -G10bulin 5A = Cl“ Albumin éé = C14 -Globulin 33 = Cl” ~Globulin 211 FIGURE 2. PHOTOGRAPH OF PAPER EIECTROPHORESIS PATTERNS OF RABBIT SERUM AND PROTEIN FRACTIONS ISOLATED FROM THE SERUM. 212 3 ‘MM Itfl£ 6“,“‘IF 3 ' o .1. v 3“. M'L.‘TQ\bI~|m Q g = p-L.;‘oa\obu\|n J '. Alb: 'm...|' 5...... m...“- FIGURE 3. PHOTOGRAPH OF PAPER ELECTROPHORESIS PATTERNS OF RABBIT ‘WHEY AND PROTEIN FRACTIONS ISOLATED FROM THE'WHEY. 213 TABLE 3. FREE LEUCINE LEVELS IN THE SERUM OF Xu35 (LACTATING RABBIT 13TH~17TH DA S POSTPARTUM INJECTED WITH 11.5 mg. OF DIP ). LEUCINE~2~C hrs. post 103. mg. % 0016 2090 2 2.55 6 2.64 24 2.50 #8 2.60 72 2.54 + 1 AV. 2.60 - 0.0534 1The mean value with standard error. *5 1 i V TABLE 4" . VAVEFACE 0F 1; 274' TEN VALUES WITH STANDARD ERRORS FOR THE PERCENT OF LEUCI I“ AI‘ID GLUTATIIC ACID IN RABBIT SERIIM AND MILK PROTEINS. Protein ‘70 leucine ‘36 Glutamic Acid Albumin 9.5 i 0.0068 17.0 i 0.015 cosine-Inns 11.7 t 0.0094 19.9 i 0.021 B-Globulins 7.3 2‘ 0.010 13.8 if 0.027 Y-mGlo'bulins 13.7 i 0.0082 10.4 1'.“ 0.014 Casein 10.0 t 0.011. 72.0 t 0.023 00.12.21.115me 11.0 1' 0.0062 17.0 .1” 0.0099 8 «lac-toglobulin 15.0 ‘i 0.0043 20.1 1 0.01.5 Immme Globulins 13.3 15 0.018 10.2 35 0.0048 "BlQOd" Serum Albumin 9.7 i”. 0.039 16.7 1‘ 0.043 215 TABLE 5. THE RADIOACTIVITY OF URINE FROM 1-35 (LACTATING RABBIT 13TH- 17TH DAYS POSTPARTUM INJECTED'WITH 45.0 UC. 0F DL—LEUCINE-Z-Clu). hrs. post inj. ml. urine counted 0PM; CPM/ml. 0.16 0.5 642 1284 2 1.0 527 527 6 0.6 145 252 12 1.2 22 18 24 1.0 --- --- 36 1.0 --- --- 48 1.0 -.. --- 72 1.0 --- --- 1The counts per minute were not significantly different from background after 12 hours post injection. 216 ABLE 6. SPECIFIC ACTIVITIES OF BLOOD AND MILK PROTEIN FRACTIONS FROM A LACTATING RABBIT (NO. x 18. 10TH-13, DAYS POST- PARTUM) INJECTED WITH 0.31 pc. (400 mg.) C1 LABELED flruGLOBULINS hrs. post. inj. DPM mg. protein counted pc./mg.:x 10)+ ”—— Blood Albumin .. _ _ 2 1:,1'4'6 @02 001.66 6 978 1‘50 O O o 098 12 965 47.8 0.091 24 792 44.9 0.076 48 647 38.9 0.075 72 460 30.5 0.068 Blood OC-G'lobul ins 2 822 40.6 0.091 6 696 36.7 0.085 12 618 32.4 0.086 24 639 39.8 0.072 48 555 41.5 0.060 72 448 35.3 0.057 Blood 8 e-Gl obulins 2 735 40.0 0.083 6 954 45.5 0.095 72 836 42.3 0.087 24 864 46.9 0.085 48 630 36.7 0.077 72 620 39.8 0.070 Casein From Milk 2 1,122 45.2 0.112 6 1,062 49.0 0.098 12 935 45.7 0.092 24 939 50.4 0.084 48 841 47.3 0.080 72 811 47.8 0.077 Milk B -lactoglobulin 2 648 49.9 0.058 6 384 21.8 0.079 12 646 45.5 0.064 24 600 45.9 0.059 48 462 40.6 0.051 72 15 38.7 0.060 217 TABLE 6. (CONT.) SPECIFIC ACTIVITIES OF BLOOD AND MILK PROTEIN FRACTIONS FROM A LACTATING RABBIT (N0. X-18, 10TH-1 TH DAYS POSTPARTUM) INJECTED WITH 0.31.pC. (400 mg.) C 4 LABELED Y -CIOBULINS hrs. post inj. DPM mg. protein counted pc./mg. x 108 Nfllkflb(-Iacta1bumin 2 720 ”509 0.071 6 811 45.7 0.080 12 776 49.1 0.071 24 669 47.5 0.06“ 48 558 46.5 0.054 72 544 47.4 0.052 218 TABLE 7. SPECIFIC ACTIVITIES OF BLOOD AND MILK PROTEIN FRACTIONS FROM A LACTATING RABBIT (NO. 11-19, 10111—13 DAYS POSTPARTUM) INJECTED wITH 2.0 pc. (800 Mg.) 0F 0 IABELEDY-GIOBULINS hrs. 1." post inj. DPM mg. protein counted pc./mg. x 10 Blood Albumin 2 978 39.5 0.112 6 762 50.2 0.069 12 1,008 45.0 0.101 24 815 41.3 0.089 48 972 48.6 0.090 72 921 50.0 0.083 Blood O(-Globulins 2 670 29.3 0.103 6 1,369 40.0 0.154 12 1,152 44.8 0.116 24 855 38.5 0.100 48 835 42.2 0.089 72 648 35.6 0.082 Blood 3 -Globu1ins 2 342 26.0 0.059 6 696 47.6 0.073 12 378 26.1 0.065 24 477 35.2 0.061 48 520 40.8 0.058 72 430 38.9 0.050 Casein From Milk 2 1,074 40.9 0.118 6 1,410 48.7 0.130 12 912 42.0 0.098 24 911 45.5 0.090 48 896 47.8 0.085 72 744 44.4 0.076 Milk B -Lactoglobulin 2 398 9.7 0.185 6 254 5.3 0.216 12 253 6.0 0.190 24 334 8.4 0.179 48 284 7.5 0.171 72 320 8.8‘ 0.164 219 TABLE 7. (CONT.) SPECIFIC ACTIVITIES OF BLOOD AND MILK PROTEIN FRACTIONS FROM A lACTATING RABBIT (NO. x.19, 10TH-13TH DES POSTPARTUM) INJECTED WITH 2.0-pC. (800 mg.) OF C LABELED Y-GLOBULINS hrs. post inj. DPM. mg. protein counted pc./mg. x 10’+ Milk:0(-lactalbumin 2 954 46.1 0.093 6 636 46.5 0.062 12 945 40.1 0.106 24 696 44.3 0.071 48 635 42.0 0.068 72 635 45.8 0.063 220 TABLE 8. SPECIFIC ACTIVITIES OF’EEOOD AND MILK PROTEIN FRACTIONS FROM A LACTATING RABBIT (NO. X-17, 10 -13TH DAYS POSTPARTUM) IN- JECTED WITH 5.16 p0. (2.0 gm.) Cl LABELED ALBUMIN. hrs. 1+ post inj. DPM mg. protein counted pc./mg. x 10 Casein From Milk 2 1,170 41.4 0.127 6 1,130 46.3 0.110 12 1,091 45.5 0.108 24 960 40.9 0.106 48 905 44.3 0.092 72 935 49.0 0.086 Milk B -Lactoglobulin 2 754 33.9 0.125 6 583 26.0 0.101 12 759 30.0 0.114 24 840 35.5 0.106 72 705 36.1 0.088 Milkx-Laotalbmnin 2 960 40.9 0.106 6 213 8.8 0.109 12 906 35.9 0.113 24 630 33.6 0.085 48 714 43.4 0.074 72 840 38.4 0.099 Milk Immune Globulins 2 725 40.0 0.082 6 1,230 44.6 0.124 12 866 38.7 0.101 24 910 42.5 0.096 48 831 45.8 0.084 72 668 39.0 0.077 Blood Y-Globulins 2 311 11.3 0.124 6 380 14.4 0.119 12 366 18.8 0.088 24 524 27.4 0.086 48 368 22.5 0.074 72 470 30.0 0.071 TABLE 8. (CONT.) SPECIFIC ACTIVITIES OF BLOOD AND MILK PROTEIN FRACTIONS FROM A LACTATING RABBIT (N0. X-l7, lOTH-l EH DAYS POSTPARTTm) INJECTED WITH 5.16 p0. (2.0 gm.) C LABELED ALBUMIN. hrs. post inj. DPM mg. protein counted jam/mg. x 10’“L Blood Cx -Globulins 2 465 27.4 0.077 6 546 25.7 0.096 12 701 35.7 0.089 24 535 31.2 0.077 48 410 26.0 0.071 72 549 38. 5 0.064 Blood 3 -Globulins 2 643 26.1 0.112 6 840 31.4 0.141 12 780 35.5 0.099 24 816 40.6 0.088 48 591 32.3 0.083 72 670 41.0 0.074 221‘ 222 TABLE 9. SPECIFIC ACTIVITIES OF BLOOD AND MILK PROTEIN FRACTIONS FROM A LACTATING RABBIT (NO. x-2o, 10TH-13TH DAYS POSTPARTUM) INJECTED WITH 13.2 pC. (3.0 gm.) OF C14 LABELED ALBUMIN. hrs. post inj. DPM mg. protein counted pc./mg. protein.x 10“ Blood'Y'-Globulins 2 1,152 41.4 0.126 6 1,020 35.1 0.131 12 1,219 45.6 0.119 24 885 39.8 0.101 48 870 40.0 0.098 72 1,014 49.7 0.092 Blood 0( -Globu_1ins 2 1,074 22.1 0.219 6 930 38.1 0.109 12 784 32.5 0.110 24 626 29.6 0.095 48 711 36.4 0.088 72 785 42.9 0.082 Blood ,8 -Globulins 2 1,296 23.8 0.245 6 1,170 46.4 0.114 12 830 39.0 0.096 24 582 35.6 0.075 48 666 42.3 0.071 72 626 48.7 0.0 58 Casein From Milk 2 762 46.0 0.0750 6 984 44.7 0.0990 12 866 48.1 0.0812 24 875 49.5 0.0796 48 641 49.9 0.0580 72 421 47.3 0.0401 Milk Immune Globulins 2 275 10.4 0.119 6 1,010 36.7 0.124 12 623 24.8 0.113 24 763 39.5 0.087 48 750 41.1 0.082 72 774 45.3 0.078 TABLE 9. (CONT.) SPECIFIC ACTIVITIES OF BLOOD AND MILK PROTEIN FRACTIONS FROM A LACTATING RABBIT (NO. X—ZO, 10TH-13TH 223 DES POSTPARTUM) INJECTED WITH 13.2 go. (3.0 gm.) OF C LABELED ALBUMIN. hrs. 4 post inj. DPM mg. protein counted pc./mg. protein 2: 10 Milk B -Lactoglobulin 2 786 30.2 0.117 6 328 11.0 0.134 12 724 29.9 0.109 24 755 35.8 0.095 48 541 30.5 0.080 72 710 39.0 0.082 bfilk:O(-Iactalbumin 2 390 21.7 0.081 6 526 25.0 0.095 12 577 29.9 0.087 24 328 20.5 0.072 48 356 24.3 0.066 72 380 27.2 0.063 224 TABLE 10. SPECIFIC ACTIVITIES OF BLOOD AND MILK PROTEIN FRACTIONS FROM A LACTATING RABBIT (N0. x-24, 10TH-11TH DA S POSTPARTUM) INJECTED WITH 1.63‘flC. (492 mg.) C LABELED O(-GLOBULINS hrs. post inj. DPM mg. protein counted ‘pc./mg. proteins x 105 Blood0(-Globulins 0.16 107,100 25.6 188.000 2 79,150 20.2 177.000 6 107,250 30.3 160.000 12 65,950 20.3 146.000 24 35,400 13.1 123.000 Blood Albumin 2 609.0 51.6 0.531 6 500.0 «46.3 0.487 12 954.5 51.3 0.838 24 543.0 49.4 0.496 Blood Y-Globulins 2 362.0 38.1 0.428 6 363.9 22.2 0.779 12 1,046.0 32.0 1.472 24 771.5 47.6 0.725 Blood 8 —Globulins 2 938.0 44.6 0.946 6 1,868.0 39.4 2.019 1.2 1,683.0 3305 2.285 24 639.5 27.2 1.059 Milk Immune Globulins 2 328.0 50.5 0.293 6 510.2 48.4 0.477 12 1,478.5 50.3 1.324 24 687.0 51.6 0.599 Casein 2 1,122 52.2 _ 0.969 6 1,416 53.2 1.199 12 1,008 51.1 0.884 24 714 51.0 0.631 225 TABLE 10. (CONT.) SPECIFIC ACTIVITIES OF BLOOD AND MILK PROTEIN FRACTIONS FROM A LACTATING RABBIT (N0. x.24, 10TH-11 H DAYS POSTPARTUM) INJECTED WITH 1.63‘pC. (492 mg.) C1 LABELED o< -CIOBULINS hrs. post inj. DPM mg. protein counted pc./mg. proteins 3: 105 Milk q-Lactalbumin ." 2 354.0 36.5 0.437 6 260.5 25.1 0.514 12 297.5 21.7 0.617 24 400.0 9.2 1.950 Milk 8 -Lactoglobulin 2 6011 49 . 6 0 . 564 3 906 32.2 1.270 1: 816 46.0 0.802 '34 449 48.3 0.419 . ,,, .1-.-p --p.o-.——.—-._—.. TABLE 11. 226 SPECIFIC ACTIVITIES OF BLOOD AND MILK PROTEIN FRACTIONS FROM A LACTATING RABBIT (NO. L27, lOTH-ll INJECTED WITH 2.37‘pC. (398 mg.) OF C ER DAYS POSTPARTUM) LABELED ,8 -CIOBULINS hrs. pOSI .. inj o DPM mg. protein counted pc./mg. protein.x 105 88mm 28mm 28mm 25mm 2:83oxn3 366,900 289,500 220,000 238,500 174,000 122.0 4920 884.0 1,067.0 424.0 716.0 1,309.0 434.0 665.5 1,271.0 918.0 548.0 2:934 4.338 5.478 4,626 420.0 3WJ5 676.0 714.0 Blood B -Globulins [+5.2 46.7 42.3 50.5 53.9 Blood Albumins 53.1 49.9 50.9 49.4 Blood.Y'-Globulins 40.0 41.0 54.5 24.7 Blood o< -Globul_ins 32.8 28.6 40.3 10.0 Casein 48.5 53.7 51.4 50.7 Milk Immune Globulins 52.5 45.9 43.4 48.5 366.000 278.000 234.000 212.000 145.000 0.104 0.409 0.784 0.939 0.478 0.788 1.080 0.794 0.915 2.010 1.030 2.460 2,720 3.640 3.930 3.580 0.361 0.364 0.703 0.708 TABLE 11. (CONT.) 227 SPECIFIC ACTIVITIES OF BLOOD AND MILK PROTEIN FRACTIONS FROM A LACTATING RABBIT (NO. X-27, 10TH-11TH D C V 4 LABELED B -CIOBULINS S POSTPARTUIvI) INJECTED WITH 2.37 110. (398 mg.) OF hrs. post inj. DPM mg. protein counted ,pc./mg. protein x 105 O(-1actalbumin 2 27601 3104 09396 6 199.0 15.2 0.590 12 242.0 14.4 0.758 24 440.0 20.0 0.991 B -Laotoglobulin 2 1,008 34.8 1.310 6 1,062 17.7 3.520 12 1,704 33.1 2.320 24 1,188 37.7 1.420 228 1,0004% x Bloodo(-Globulins ‘ Blood YC-Globulins ° Immune Globulins From Milk :p___x________x___ ~——x ________________________———_‘i 10000 "’ V) C) ‘\ x E“ F 10.0 — 9L 05 o \ O O 1.0 .— / 0.1 1 1 l 1 0 2 6 12 24 hrs. post inj. FIGURE 4. THE LOG OF SPECIFIC ACTIVITY (pm/mg. x 105) vs. TIME (hrs. post inj.) FOR BLOOD o<-CLOBULINS, BLOOD Y-CLOBULINS AND IMMUNE GLOBULINS FROM MILK ISOLATED FROM x..24 (LACTATING BIT 10TH—11TH DAYS POSTPARTUM INJECTED WITH 1.63 ,UC. OF C1 LABELED OA-GLOBULINS). 229 1,000.0 '- x Blood )3 -Globu1ins 0 Blood Y -Globulins o Immune Globulins From Milk /.- \x 100.0 " /03 ,uc/mg x /05 H O °o r 1.0 — 01° ./ 001 1 1 l I 0 2 6 12 24 hrs. post inj. FIGURE 5. THE LOG 0F SPECIFIC ACTIVITY (pc./mg. x 105) VS. TIME (hrs. post inj.) FOR BLOOD p -CLOBULINS, BLOOD Y—GLOBULINS AND IMMUNE GLOBULINS FROM MILK ISOLATED FROM X—27 (LACTATING BIT 10TH-11TH DAYS POSTPARTUM INJECTED WITH 2.37 p0. OF C1 LABELED p -GLOBU‘LINS). ”31:51".- “UL”; USE 0?!” r 3.. .1” A‘ 3 .‘« ' “ ‘1' ‘7' I ’ 54‘1“."