GENERAL METABOLISM 02* COBALT STUDIED IK CHICKENS AND DOGS By Lee Cheng»Chun A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements of the degree of DOCTOR OP PHILOSOPHY Department of Physiology and Pharmacology 1952 ProQuest Number: 10008279 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10008279 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 j ' Co * S b ACKNOWLEDGMENTS The author wishes to express his appreciation to Dr. Lester P. Wolterink for his encouragement, guidance and advice during the course of these experi­ ments and in the preparation of this manuscript. Acknowledgment is also due to Dr. B. Y. Alfredson for his encouragement and for his aid in pro­ viding facilities in the department; and to Drs. W. D. Collings, E. P. Reineke, J. Meites and C. P. Huffman for their interest and suggestions. Many thanks are also due to Mr. W. Priedberg, Mrs. E. P. Monkus and Mr. R. L. Cornwall for their assistance in the preparation of the experi­ mental animals; and to Mr. J. Monroe for caring for the animals used in these experiments. Pinally, the author wishes to express his gratitude to the Division of Biology and Medicine, U. S. Atomic Energy Commission, for the funds tinder which this work was done. THESIS ABSTRACT Although specific cobalt deficiency diseases have been described in ruminants from many parts of the world, little is known with respect to the passage of cobalt through the living system* Experiments were set up to study the general metabolism of cobalt particularly in non-ruminants (chickens and dogs). Particular emphasis was placed on the role of the intestinal tract, of the renal tubules, and of the liver. A radioactive isotope of cobalt, Co^, was used. Cobalt 60, as Co SOI4., *s Both readily absorbed from the intestinal tract and Msecreted" or "continuously diffused" into the intestinal tract through In its wall in both the chicken and the dog. the chicken, most of the C o ^ found in the caecumenters from gut.In the dog, the caecum In the plays an insignificant role. the chicken, Co u is greatly concentrated in the caecal contents, in the caecal wall, and in the large and small intestines regardless of the route of administration. bile of the chicken. A very small amount of Co^® is found in the bladder C r\ In the dog, the highest concentrations of Cow are found in the liver, in the intestinal wall and in the contents of the first part of the jejunum, in the kidney, in the gall bladder bile and in the bladder urine. The spleen contains a very low amount of Co 60 . Cobalt 60 passes into the extracellular fluid soon after intravenous injection of its inorganic form in the dog. Blood cells contain an insig­ nificant amount of Co^. In the dog, the plasma Co^° quickly filters through the glomeruli follow­ ing a single intravenous injection. The maximal values for the rate of urinary excretion are reached within one-half to three hours. On the other hand, the rate of biliary Co^O excretion reaches a maximum about fire to seven hours after injection. In dogs receiving a constant infusion, relatively constant plasma Co concentrations are maintained. 60 The rate of urinary Co excretion in these dogs remains constant but at a level dependent upon the rate of infusion. However, the rate of biliary excretion increases gradually and reaches a plateau at about four to seven hours. 60 The renal clearance of Co within three hours after intravenous < • injection averages 27 ml. per minute per square meter of surface area. About three-fourths of the filtration load is reabsorbed by the tubules. In acute experiments of long duration (up to thirteen hours), renal clearances of Co^® decline due to an increase in the tubular reabsorption. In the dog, the half-time for the removal of Co^® from the blood is 60 20$ faster when injected as the cysteine-Co complex than when injected as Co^®S0ty. The transfer of Co^® from the peritoneal cavity to the blood is 3*3 times faster when injected as the cysteine-Co^® complex than when injected as Co 60 SOty. A form (or forms) of Co° the bile and in the urine. other than its inorganic form is found in The Co 60 in these samples is reabsorbed from the gut of young chicks at a considerably faster rate than is inorganic CONTENTS INTRODUCTION .................................. REVIEW OP LITERATURE The Physiological Punction of C o b a l t ................. History and Importance of Cobalt in the Nutrition of Ruminants............................ . Probable Mechanism of Cobalt Action and Its Relation to Vitamin B i g .................................. Metabolism Studies of Cobalt ...................... The Physiology of the Caecum in the P o w l ............. Renal and Hepatic Clearances . . . . * ............. . Renal Clearance .................................. Hepatic Clearance ................................ METABOLISM OP COBALT 60 IN C H I C K E N S ........... ......... A. Methods ......................................... B. Results ......................................... Trial I, Large Intestine Not Ligated . ............. Intestinal Recovery . . . . .................... Extracellular Pluid R e c o v e r y ................... Caecal Recovery...................... . Trial II, Large Intestine Ligated ................. Intestinal Recovery ............................ Extracellular Pluid R e c o v e r y ................... Caecal Recovery . . . .......................... Ratio to Blood ................................ Comparative Data from Both T r i a l s ................. C. Discussion ....................................... METABOLISM OP COBALT 60 IN D O G S ................................. A. Methods ................................................... General Remarks . - ..................................... Distribution of Co in Blood ........................... Rate of Co Absorption from Peritoneal Cavity and Its Rate of Elimination from Blood When Injected In t r a p e ritoneaLly.............. .................. Reabsorption of Co from the Urine and Bile Samples . . Renal Clearance, Tubular Reabsorption and Hepatic ............................. Clearance of Co®^ Tissue Distribution of Co in Dogs . . . . . .......... B. R e s u l t s .......... .............. .......................... Distribution of Co in Blood . . . . . . . . . . . . . . Rate of Co®^ Absorption from the Peritoneal Cavity and Its Rate of Elimination from Blood When Injected Intraperitoneally ................................. Reabsorption of Co from the Urine and Bile Samples . . Renal Clearance, Tubular Reabsorption, Hepatic Clearance, BateSggf Urinary Excretion and Biliary Excretion of Co ....../■ * . . • • » • • • » • • • • • Tissue Distribution of Co in Dogs . . . . ............ 0. Discussion....... ..................................... Distribution of Co®® in Blood and Probable ProteinBound F o r m s .................... . . ............. Urinary and Biliary Excretion of Intravenously Injected Inorganic Cow . . . . , . Renal Clearance and Tubular Reabsorption of Go®® . . . . . Tissue Distribution, Intestinal Absorption and "Secretion” of C o ® ® ....... ..................... SUMMARY ................................................... BIBLIOGRAPHY ............................................... APPENDIX 1* The Co®® Mass Absorption Coefficient . ................. 2. Methods and Calculations for the Study of Renal Function in the Dog ......... ................. 3. Concentration of Co in Whole Blood, Plasma, Protein-free Plasma Filtrate and Relative Distribution of Co®® in B l o o d ........... ................. - .............. k. Renal Clearances of Creatinine, PAH and Co°® in Sodiumpentobarbltal Anesthetized Dogs of Short Duration (up to 3........ .......................... fa .............. 5. Renal Clearances of Creatinine, PAE and CoDU in Sodiumpentobarbital Anesthetized Dogs of Long Duration (up to 12 hours)....................................... - . 6. Blood Concentration, Urinary and Biliary Excretion of Co in Dog No. 1 ................................... .. 7. Blood Concentration, Urinary and Biliary Excretion of Co®° in Dog No. 2 ................................... ^ . 8. Blood Concentration, Urinary and Biliary Excretion of Co in Dog No. 3 ................................... - . 9. Blood Concentration, Urinary and Biliary Excretion of Co®® in Dog No. U ............... .......... ............ 11. Blood Concentration, Urinary, Biliary and Total Excretion of Co in Dog No. 6 ................................ 12. Blood Concentration, Urinary, Biliary and Total Excretion of Co in Dog No. 7 ................................ 13. Blood Concentration, Urinary, Biliary and Total Excretion of Co in Dog No. 2*4-................................ l^f. Blood Concentration, Urinary, Biliary and Total Excretion of Co®° in Dog No. 25 ............................ 14. Tissue Distribution of Co®® in Two Dogs Twelve Hours after Injection . . . . 1 INTRODUCTION The metabolism of cobalt, present in trace amounts in the tissues of animals and plants and in the cells of microorganisms, has attracted much attention in the last two decades. Interest was first aroused by the find­ ings of Ulmer and Underwood (1935). Marston (1935) 80(1 Lia«s (1935) * that "enzootic marasmus" and "coast disease", of common occurrence in areas of the West Australia and the South Australia, were due to cobalt deficiency. At the present time, cobalt is known to serve no functional purpose in the animal body other than as an integral part of the accessory food factor, vitamin B-^g, and as an agent to induce erythrocytosis. Our present knowledge of the metabolism of inorganic cobalt is illustrated diagrammatically in Figure 1, As indicated in the diagram cobalt is probably lost from the body by three routes: in the feces, in the urine and in the bile. Fecal loss constitutes a large fractionof the dose when administered orally or when introduced directly into the stomach. Intravenously injected cobalt, on the other hand, is chiefly excreted through the urine. partially included in the fecal cobalt. Biliary loss is at least It represents that fraction of the cobalt which is carried through the liver and returned to the intestinal tract in the bile. Whether some of this can be reabsorbed subsequently does not appear to have been investigated. In view of the lack of knowledge concerning the passage of cobalt through the living system, it becomes highly desirable to investigate: 2 1 . what proportion of administered inorganic cobalt is tied up in the intestinal tract when it is given either orally or intravenously; 2. the relative amounts of cobalt excreted through the intestinal tract and through the kidneys following the two different routes of administration; 3 . partitioning of cobalt between the fluid compartments of the body; the rates of biliary and urinary excretion of cobalt in relation to the blood cobalt level; 5* the physiological role of the tubules of the kidney in handling this trace element; 6 . the different turnover rates for inorganic cobalt and amino acid cobalt complexes in the animal body; 7 . the reabsorbability of the forms of cobalt found in bile and in urine. The chicken is a favorable experimental animal in which the first two problems may be investigated, because large numbers are readily available and because the surgical techniques are convenient. Since the caeca of birds are believed to be the main site for the synthesis of various accessory food factors which are the by-products of the metabolism of their bacterial popu­ lation, emphasis was also placed on these structures. Determinations were made of the amount of cobalt "fixed” in the entire caeca, or in the caecal wall versus the caecal contents, both when these organs were free and when they were tied off at their junction with the intestine. The dog was chosen as the experimental animal to be employed in the studies of the next four problems, due to its large body size and its relative­ ly high rate of urine and bile flow. In order to compare the metabolic role of cobalt in the intestines with that in the chicken, inorganic cobalt was also injected intravenously or introduced directly into a loop of small 3 intestine of the dog. Various segments of the intestinal tract and tissues were analyzed for cobalt distribution. Por the last of the problems listed, the reabsorbability of cobalt in the urine and bile samples collected from the experimental dog was studied by determining the rate of their intestinal absorption in three-day-old White Leghorn chicks. Preliminary attempts to identify the cobalt compounds present in these samples were made using paper partition chromatographic and radioautographic techniques. DIAGRAM s OF COBALT CIRCULATION s /. IN THE SC: BODY. □ FIGURE 1+ 5 REVIEW OF LITERATURE The Physiological Function of Cobalt History and Importance of Cobalt in the Nutrition of Ruminantst Certain peculiar diseases among grazing ruminants have been known for many years and all over the world. Animals suffering from these diseases lose appetite and weight, become weak and anemic, and finally die. Each disease has been recognized as being limited to a certain area of the world and prevention and cure can be achieved by transferring animals from one locality to another. All these diseases have different names but similar symptoms in different parts of the world, and now appear to have the same etiology. As summarized by Beeson in the United States (1950) by Marston in Australia (1952), among these disorders are "bush sickness11 and "Horton Main disease" of Hew Zealand, "enzootic marasmus" of Western Australia, "coast disease" of South Australia, "pining disease" of the Cheviots and of other parts of Scotland, and of Devon, Cornwall, Herefordshire and Worcester­ shire of England, an unnamed disease in Dartmoor of England, "nakuritis" of Kenya Colony in East Africa, "salt sickness" of Florida, "neck ail" of Massachusetts, "Burton-ail" of Hew Hampshire, "Grand Traverse disease" of northern Michigan, "ailments" of Wisconsin, and similar diseases in Canada, Hebrides, Ireland, Horway, Denmark, Sweden and Estonia. There is little doubt that these diseases seriously menace the ruminants in other sections of the world where there is a shortage of cobalt in fodder plants. The development of the studies on these maladies of grazing animals has been summarized by Marston et al. (1938), and discussed extensively by 6 Wunsch in New Zealand (1937* 3-939) where the studies on "hush sickness” began a half century ago. In view of the regional distribution of these ailments and of the curative effect of shifting animals from "sick” to "healthy” areas, it has been hypothesised that the nature of the soil, and thus of the forage, in the affected regions was involved in producing these nutritional disturb­ ances among ruminants. The first publication in this series appeared in 1911, and was cited and summarized in 192h by Aston (192h), who concluded that the disease was due to iron deficiency, and could be cured and pre­ vented by administration of ferric ammonium citrate. Although he later claimed to have confirmed his conclusion (1932), further studies made in the North Island of New Zealand (Grimmett and Shorland, 193*0* and in South Island (Bigg and Askew, I93U 5 1936) produced no evidence to support the curative treatment of iron salts. The discovery of cobalt as the deficiency element was made independently by two groups of investigators. Ifilmer (1933) reported that "enzootic marasmus” in West Australia and similar diseases are due to a shortage of some mineral essential for iron metabolism and associated in nature with the latter. Later, he and Underwood obtained an iron-free extract of limonite curative for "enzootic marasmus" (193*0 » a&d identified the curative agent as cobalt (1935). In the meantime, initiated by the fact that animals with "coast disease” in South Australia suffer from anemia and that cobalt produces polycythemia in rats (Waltner and Waltner, I929), Marston (1935) and lines (1935) succeeded in the treatment of this disease with cobalt. Their reports in great detail were reviewed later (Marston et al., I93S). 7 Following the reports of successful use of cobalt in Australia, cobalt salt6 were demonstrated to be effective In curing and preventing similar diseases in the South Island of New Zealand (Askew and Dixon, 1936) in the North Island (Wall, 1937)* in Florida of the United States (Neal and Ahmann, 1937). in Dartmoor of England (Patterson, 1937* 1938). ia western Canada (Bowstead and Sackville, 1939)* in the Cheviot Hills (Corner and Smith, 193*0 and in Cornwall and Devon of Scotland (Patterson, 19^-6). The earlier concepts in regard to cobalt metabolism and its relation to ruminant nutrition have been reviewed by three groups of investigators (Huffman and Duncan, 19^? McCance and Widdowson, 19^4; Bussell, 19*&). The present status of knowledge of this trace element in the nutrition of animals 1 and plants has been summarized by Marston (1952). The role of vitamin B-^g, an Important, biological compound of cobalt, in animal nutrition has been reviewed by Smith (1950-1951)? its clinical aspects by Ungley (195I). Probable Mechanism of Cobalt Action and Its Helatlon to Vitamin Bip: The suggestion that cobalt exerts its action, in the ruminant animal, either within the lumen of the alimentary tract or during its passage through the wall is based upon three pieces of evidence. tial for the nutrition of ruminants. First, cobalt is only essen­ Other herbivorous animals have not been shown to be affected by low cobalt pastures on which sheep and cattle develop acute deficiency (Marston et al., 193®)* Bats have been shown to be unaffected when intake of cobalt is less than 0.6 microgram of cobalt per day (Underwood and Elvehjem, 1938) , or less than 0.3 microgram of cobalt per day (Houk et al., I9U6 ). Bats raised on rations which provided between 0.15 and 0.35 microgram of cobalt per day for three generations, grew as normally as those which received a supplement of 10 micrograms of cobalt daily (Marston* 19^9). Babbits remained healthy when they received less than 0.1 microgram of cobalt per day (Thompson and Ellis, 19^7). Thus, non-ruminants require cobalt only in extremely small quantities, if a£ an. when administered orally. the animal. Second, cobalt is effective only Parenterally injected cobalt is not beneficial to This suggests that cobalt functions through some mechanism in the alimentary tract. McCance and Widdowson (19*&) were the first to indicate the ineffectiveness of parenteral administration of cobalt and other groups confirmed it (Becker et al., 19^9* Becker and Smith, 19^9* 1951* Marston and Lee, 19^9? Gall et al., 19^9; Smith et al., 1950)* When injected intra­ venously, the cobalt concentration in the blood and tissues might reach a level as high as ten times above that usually found in normal sheep without any beneficial effect to the animal (Marston, I9U9 ; Marston and Lee, I9U9). Third, the cobalt must be administered frequently. Marston (19^9) reported that sheep on cobalt-deficient pastures, given orally a supplement of cobalt of 1 mg. per day thrice weekly, remained in normal health. However, correspond­ ing doses administered at intervals of six weeks could not prevent the fatal malady and larger doses given at intervals of two weeks failed to maintain normal health. Microorganisms which normally flourish in the alimentary tract have been known to be important in the nutrition of the ruminant (Marston, 19^9* Marston and Lee, 19**9). Tosic and Mitchell (19^-S) reported that orally administered cobalt is taken up and concentrated by rumen microorganisms. The nature and the numbers of rumen microorganisms have been observed to be directly influenced by cobalt intake (Gall at al., 19^8? 19^9? Gall and Huhtanen, 1950). Thus, the function of cobalt in the ruminant might relate to the nature 9 and. activity of the mixed population of microflora. The cellulose-splitting microorganisms of the rumen were found to he not markedly affected in the cohalt-deficient lamh (Becker and Smith, 19*19} 1951)* as indicated hy the fact that the efficiency with which crude fiher is fermented within the digestive tract is unimpaired. A low-cohalt diet might then affect only the bacteria other than the cellulose-splitting microorganisms, or it might influence the bacteria hy preventing the synthesis of important food factors which are normally required in the balanced nutrition of the animals. The essential nature of cobalt for the synthesis of B vitamins by the rumen microorganisms has been suggested (Bay et_ al., 19*49); however, combined treatment with various B vitamins failed to improve the syndrome of cobalt deficiency in lambs (Bay et al., 19*49). However, with similar treatment, a favorable result was later claimed by the same group of investigators (Hale et al., 1950b). Investigators have shown earlier (Filmer, 1933? Marston, 1935) that some factor in liver has therapeutic value in the treatment of the ruminant malady. However, the slight response of cobalt-deficient sheep to continued therapy with liver extract is only temporary (Marston, 19*49). It is clear now that the dosage used was too small to meet the requirement of the cobalt-deficient sheep. Subcutaneous Injection of 15 U.S.P. unite of a purified liver extract daily has been reported to be effective in the treatment of cobalt deficiency in lambs (Becker and Smith, 1951). The absence of response when the same dosage of liver extract was administered orally in the same experiment might be the result of destruction of its activity within the alimentary tract or of inefficient absorption from the tract. It is evident that some factor or group of factors contained in the liver extract possesses curative activity which prevents the onset of the syndrome produced by a cobalt deficiency. 10 A comparison of the rate of response of cobalt-deficient lambs to oral administration of cobalt and to subcutaneous injections of a purified liver extract have been made (Becker and Smith, 1951). An immediate response to the liver therapy indicates that the liver extract is a direct supply of the accessory food factor or factors required by the deficient lambs. On the other hand, a latent period is essential for the microorganisms normally living in the alimentary tract to synthesize the deficient metabolite or metabolites following the stimulation of cobalt therapy. With the recognition of vitamin B^g (Bickes et al., 19^8a; Smith 19^Sa), which is a cobalt-containing complex (Hickes et al., l9^Sb; Smith, 19^8b), it was suggested that the syndrome of cobalt deficiency originates not from a lack of cobalt per se, but from a shortage of vitamin B^g. This belief was immediately strengthened by the important finding that the rumen contents are normally rich in vitamin B^g (Gall et al., 19^8; Hale e£ al., 1950a; Lewis elfc al., 19^9) . and that the synthesis of this vitamin in the ali­ mentary tract is seriously impaired when the concentration of cobalt in the ingesta falls below the level necessary to secure a balanced nutrition of the animal (Abelson and Darby, 19^9* G®11 et al., 19^8; Hale et al. , 1950a). However, attempts failed to correct the deficiency symptoms by supplementation with pure vitamin B^g, either per os, or parenterally, in sheep (Marston and Lee, 19^9)* lambs (Becker and Smith, 19^9* 195*» Becker et al., 19^9)• It is now clear that the dosages employed in these experiments were insuffi­ cient to meet the nutritional demands of the ruminant. This has been clearly demonstrated by observations with sheep (Marston and Lee, 1951). While no regression of the symptoms was obtained in sheep on cobalt deficient pastures treated for four months with 15 micrograms of vitamin Blg per week, injected 11 intravenously; the sheep responded immediately when the dosage was increased to 300 micrograms of vitamin B^2 Per week. The most outstanding work concerning the direct relationship between the metabolism of inorganic cobalt and vitamin was reported by Davis and Chow (1951) that appropriate amounts of aureomycin or the increased amount of C o ^ incorporated in the diet of rats caused an increase in Co^-labeled vitamin B^g activity. They related this effect of the antibiotic to the intestinal bacterial flora. Monroe et al. (1952b) further demonstrated that considerable biosynthesis of vitamin B-^g took place in the intestinal tract Cr\ following oral administration of Co , and also occurred in the tissues following intravenous injection. The livers and kidneys of ruminants are a very potent source of vitamin Bl2 . Beef kidney and liver have been found to contain 15 to 20 micrograms of vitamin Bj.2 Per 100 gm. wet weight (Lewis et al., 19^9) * a&d k2 to kf micrograms per 100 gQ. after tryptic or pancreatic digestion (Thompson et al.. Since ruminant livers are those chiefly used for the preparation of liver extract (Smith, 1951). there is little doubt that vitamin B^g is the curative principle present in liver extract which possesses therapeutic value for the cobalt deficiency symptoms. It Is known that routes of the intermediary metabolism are somewhat different in ruminants and non-ruminants (Marston, 1939? 19^8). The shorter chain acids produced by fermentation of carbohydrates in the rumen are the main sources of energy for ruminants. This leads to the belief that ruminants may have different requirements for the accessory food factors, including vitamin B^2 » The high vitamin B^g activity of rumen contents (Hale et_ al., 1950a; Lewis et al., 19^9), and the high concentration of this vitamin in the ruminant liver and kidney (Lewis et al., 19^9* Thompson et al., 1950) again 12 suggests that this concept is true. In view of this, it is hardly surprising that all natural and experimental syndromes of cohalt deficiency have been reported only in ruminants. Other animals may require such small quantities of this particular food factor that they usually can get enough of it from the "basal diet". Shis may explain the failure to produce experimental cohalt deficiency in rats (Houk et al., 19*1-6; Marston, 19^9; Underwood and Elvehjem, 1933), and in rabbits (Thompson and Ellis, 19*+7)• The possibility has not been entirely excluded, at the present, that other accessory food factors in addition to vitamin 3^2 Bay* ia part, play some role in the syndrome of cobalt deficiency. Metabolism Studies of Cobalts The suggestion that bile serves as a pathway for the elimination of cobalt was first made in 187** (Mayencon and Berger et) and again in 188h (Stuart). But it was not until 193^ (Caujolle) that cobalt was definitely found in the bile after parenteral administration. Greenberg et al. (I9**3b) were able to collect bile through biliary fistulae and measure its cobalt radioactivity after administration of 0 .1 mg. of radioactive isotopes of cobalt to rats. About 3*5 *♦ 1.*$ of the injected dose was recovered 72 hours after intravenous injection, but only 2 .0 * 0 .2$ after oral administration. By using the same technique, Sheline et al. (I9H6) reported a recovery of 5$ of the injected dose from the bile of five dogs at the end of 72 hours after intravenous injection of 10 to 26 micrograms of radioactive cobalt. Significant amounts of cobalt were not found in the pancreatic juice collected from pancreatic fistulae in dogs (Sheline et al., 19**6). Kent and McCane (19**1) have reported that little dietary cobalt is absorbed by man and that the kidneys are the organs responsible for the elimination of the absorbed cobalt. During the past ten years or so, radio­ active isotopes of cobalt have been used extensively in the studies of the metabolism of this tracer in the animal body. Urine has been found to be the chief pathway for the true excretion of cobalt in rats (Copp and Greenberg, I9HI; Greenberg et al., 19**3b; Gomar et_ al., 19**6a); and in cattle (Comar and Davis, 19**7»* l9**7b? Gomar et al., 19**6a; 19**6b). These authors reported that 65# of the injected cobalt in cattle and from 65 to 90$ in rftts is eventually eliminated in the urine, A large majority of it is excreted during the first hours after intravenous injection. and 5# in rats is removed in the feces. from Ho Seven to 30$ io- cattle However, after oral administration, to SO# of the dose is eliminated in the feces and from 10 to 20# in the urine in rats. In cattle, when cobalt is given orally or Introduced directly into the rumen, between 63 and SO# can be accounted for in the feces and only 0.5# in the urine. It has been reported that less than 9# of the cobalt is retained in rats four days after intravenous injection (Copp and Greenberg, I9H1) and that 5# remains in cattle ten days after injection (Comar et al., 19H6a). At the end of 72 hours, the liver retains 2.5 + 0.6# of the dose when given orally and 3 .5 ♦ 0.7# when injected intravenously in rats (Greenberg et al., 19**3*>) • After oral administration or intravenous injection, the distribution of radioactive cobalt in tissues has been studied in mice (Lawrence, I9H7); in rats (Copp and Greenberg, 19**1» Cuthbertson al., 1950? Ulrich and Copp, 1951)i in rabbits (Comar and Davis, 19**7b)» in pigs (Braude et al., 19H9; Comar and Davis, 19H7b); and in cattle (Comar and Davis, 19H7&; 19**7”b? Comar et al., 19H6a; 19H6b). Injected cobalt is generally distributed throughout the tissues, high concentrations being found in the glandular organs, Ik especially the adrenals, kidneys, thyroid, liver, thymus, intestinal lymph glands, pancreas, spleen and reproductive organs. Low concentrations of cobalt are found in muscle, bone, cartilage, white bone marrow (long bones), tongue, fat, eyes and brain. Ho cobalt has been recovered from the pituitary. A consistently higher concentration in the lymph glands might suggest the importance of the lymphatic system in the transport of cobalt (Comar and Davis, 19**7b). Small amounts can be detected in the contents of the abomasum (Comar and Davis, 19**7a); none in that of the rumen (Comar et al., 19**6b). Orally administered cobalt is poorly absorbed in mammals, and in general, the distribution in the tissues parallels that of injected cobalt. A young calf absorbs a greater percentage of orally administered cobalt than does the older animal (Comar and Davis, 19**7b). fin In comparing the tissue distribution of Coou and vitamin C o ^ in chicks, Monroe et al. (1952a) reported that vitamin labeled with is retained by the tissues to a greater extent than is C o ^ following intraperitoneal injection. The distribution of C o ^ in tissues is in general agreement with the reports from other animals except that a very low concentration is found in liver. Monroe elb al. (1952b) have recently reported evidence for vitamin B^g biosynthesis in the tissues of sheep. They claimed that more than ten times fio as much Co is converted to vitamin B^g after oral administration as after intravenous injection, and that there is apparently considerably biosynthesis of vitamin B^g tissues after intravenous injection. Their evidence sug­ gests that the adrenals and spleen are possibly the chief tissues for this internal synthesis. They found three distinct fractions containing labeled cobalt, namely a vitamin B^2-lllc®* 811 inorganic fraction and a fraction in 15 which cobalt is in some "bound" form, in various tissues. the "■bound" form is apparently lacking. In the urine, About JO to 90$ of the total Co 60 in the feces, and 30 to 30$ in the small intestine are in the vitamin Bi2-like and bound forms. About 30$ of the total blood Co^® is in vitamin B^g-Hke and bound forms seven days after oral administration of inorganic Co^, as compared with 75$ after intravenous injection. During the seven-day period, $4-.ty$ of the oral cobalt dose was recovered in the feces and 10.8$ in the urine. After intravenous injection, there was 8.0$ of the dose excreted in the feces and 77*7$ in the urine. The cobalt concentration in blood drops in a matter of minutes (Comar and Davis, 19^7b) and its total recovery in the blood falls to 1$ or less after 10 to 15 days (Comar et al., 19h6b) when intravenously injected into cattle. However, none can be detected in the blood when cobalt is introduced directly into the rumen (Comar at al., 19^6b). Very small amounts or no cobalt at all is found in the milk or in the saliva (Comar et al., 19^6b). Extremely small but definite amounts of cobalt are transmitted across the placenta for storage in the liver of the fetus when cobalt is injected or fed to pregnant cows (Comar and Davis, 19^7&)* In regard to the internal metabolism of cobalt, no Indication of species differences between rabbits, swine and cattle has been found (Comar and Davis, 19^7*0* It has been reported 60 that blood cells account for 20 to 30$ of the whole blood Co in two pigs 16 weeks old which received 2.138 mg. of radiocobalt chloride in the diet during a period of ^3 days (Braude et al., 19^9)* In weanling rats after 90 days on a diet containing 0,006 (2 microcuries) ppm. inorganic radioactive cobalt, the blood cells contained lJO to 30$ of the whole blood Co 60 . This was further increased to 70 to 80$ in the weanling rats after Ho days on diet containing 16 £r\ 0.21 microcuries of Cow in the form of radioactive yeast extract daily (Guthbertson et al.. I95O). The Physiology of the Caecum in the Fowl That digestion of protein and starch takes place in the caeca has been known since the beginning of the century through a direct study of the digestion employing a caecal fistula (Maumus and Launoy, 1901; Maumus, 1902). Crude fiber digestion has been reported to be a particular function of the caeca in chickens, as indicated by the lower digestibility of fiber in caecectomized chickens than in normal birds (Badeff). This was substantiated by the finding that the caecal content of crude fiber is always lower than the intestine content (Boseler, 1929), It has been suggested that bacterial activity in the caecum is responsible for the digestion of crude fiber (Mangold, 1928). Further work indicates that bacterial decomposition in the caecum converts the crude fiber into glucose and organic acids (Blount, 1937)• ®h© report that the total population of microorganisms in the caeca is larger than in any other part of the alimentary tract in chickens (Johanson £t al., 19^8) also suggests that a mixed population of microorganisms plays an important role in caecal digestion in the bird. It is believed that such food materials as crude fiber and certain proteins resistant to peptic and intestinal digestion are decomposed by mixed activities of microorganisms and by enzymes in the caeca of chickens. Xaupp (1919) was the first to report a neutral or slightly acid reaction in the caecum of domestic fowls. A decrease of pH in the caeca and large intestine (Olson and Mann, 1935* Farner, 19^2) might be attributed to the presence of organic acids resulting ? from bacterial decomposition of starch and proteins. Evidence has not been 17 found to support the view that any significant amount of fat digestion takes place in the caeca of granivorous fowl. Browne (1922), on the basis of the microscopic anatomy of the caeca, suggested that absorption may readily take place during the passage of Intestinal contents into and out of the caeca. By giving colored fluid orally as well as by injecting it directly into the cloaca, evidence was obtained that caeca in chickens may serve as fluid reservoirs. Since crude fiber digestion in the caeca has been reported, glucose absorption has been suggested (Blount, 1937? Mangold, 1928; Badeff). Non-protein nitrogenous substances appear to be absorbed from the caecal contents (Mangold, 1928). The presence of less crude protein but of more pure protein in the caecal contents than in the intestinal contents may indicate an absorption of amides (Boseler, I929). The synthesis of ”B n vitamins by coliform microorganisms has been claimed to take place in the caeca of chickens (Johanson et al., 19^8). Their absorption, however, is not certain. Mineral absorption has been postulated (M'Gowan, I93O). There is little doubt that water is absorbed in caeca. Thus, caecectomized chickens pass more moist feces (Mangold, I92S; Badeff), and the percentage of water in the intestinal contents is higher than that in the caecal contents (Keith et al., I927) , and chickens with ligated caeca suffer from diarrhea (Browne, 1932)* After the present experiments on the Co^® metabolism in chickens had been completed, Pandurang (1952) of this laboratory, by injecting C o ^ solution directly into the lumen of the tied caecum in chickens, demonstrated that the caecum absorbs both Co^® and water, and that Co^® is in some bound form in the caecal contents. IS The caecum is apparently not essential to fowl. The removal of both caeca in the chicken has been reported to cause no harm to health (Blount, 1937* Radeff), nor has any reduction in egg production been observed (Mayhew, 193*0 • Similar results were observed following ligation of both caeca in turkeys (Schlotthauer et al., 193*0. hauer et al., 193*0 Fertility remains unimpaired (Schlott- digestibility of the rations remains unchanged (Hunter et al., I930) in turkeys which have undergone the same operation. Therefore, the caecum is not absolutely required by fowl under normal condi­ tions. However, on minimal or deficient rations, it may assume greater importance. The caecal microorganisms might then come into the picture by either synthesizing certain essential nutrients available for their host, or competing with their host for limited essential nutrients. Browne (1922) was the first to suggest that some intrinsic mechanism within the caecal tubes controls the caeca, causing them to fill up and later to empty. The distension stimulus resulting from the filling of the caeca causes evacuation. In live chickens with a caecal fistula, when the caecum was flushed with water, the contents were found to be ejected forcibly (Pandurang, 1952). It is generally stated that contraction brought about by distension stimulus leads to emptying and that KsuctionN (due to a negative pressure created by active dilatation) results in filling (Browne, 1922} Mangold, 1928). It has been observed that the caeca empty independently of each other and of the rest of the Intestines (Mangold, 1928), and that one caecum evacuates one day and the other the next day (Boseler, 1929). A complete passage of food through the alimentary tract of chickens rarely requires more than twelve hours; however, a complete replacement of caecal contents requires about five days and one caecal discharge occurs for every 19 8even to eleven intestinal discharges, depending upon the nature of the feed (Eoseler, 1929). In testing the rate of passage of various inert materials through the alimentary tract of a variety of animals, Including human beings, Hoelzel (1930) found that most of the heavy materials are retained in the gizzard in the pigeon and chicken and in the duodenum and ileum in the chicken as well. No test materials were found in the caeca of chickens, although food materials were present. The nervous control of caecal activity is not understood. Observations have been made on young house wrens in which "reflexes'* were found associated with feeding and defecation (Heed and Heed, I925). Immediately upon swallow­ ing food, a complicated "reflex" is observed, which leads the young wren to defecate in a very unusual position in which the parent bird can easily collect the excreta as voided, and carry it away. Caeca are usually paired in fowls. caeca have been reported (Maumus, 1902): tarians} 3 However, four general types of 1. well developed caeca in vege­ 2. rudimentary caeca in carnivores; 3* only one caecum; h. no ? caecum at all. Eenal and Hepatic Clearances Renal Clearance: It was Moller, McIntosh and Van Slyke (I929) who first used the term "CLEARANCE" in connection with the excretion of urea. They defined it as the volume of blood which contains the amount of urea excreted each minute by the kidneys. This is not necessarily a real volume cleared completely but a "virtual" volume. No attempt was made to explain this clearance in terms of any particular process in the kidney. In 1931* Jolliffe and Smith 20 extended this term to the excretion of creatinine; and since then it has been generally used to describe the excretion of other substances. In his recent book, "The Kidney”, Smith (I95I) has pointed out that inulin clearance is, in all species, a direct measurement of the glomerular filtration rate. In the dog, creatinine and inulin clearances have been found to be identical under all conditions (Bichards et al., 1936; Shannon, 1935* 1936? Tan Slyke et al., 1935)* Diodrast clearance has been used to measure the "effective renal plasma flow" (Smith et al., 1938). Because of the observations that at low plasma levels sodium para-aminohippurate (PAH) clearance is identical with that of diodrast, that PAH does not penetrate the red blood cell in vivo, that it is not toxic, that it is less bound by plasma proteins than is diodrast, and that the chemical determination is simple, PAH has been extensively used in place of diodrast for the deter­ mination of plasma flow through the kidney (Chasis et al., 19^5X Goldring and Chasis, 19*&). Houck (19^8) has presented a statistical analysis of filtration rate and effective renal plasma flow in 79 aormal, trained, female dogs (516 to 7jk individual 10-minute urine collection periods in 258 simultaneous clear­ ance observations). The mean filtration rate per sq. m. of body surface area is 8^.^ ml. per minute, with a standard error of 2.2; the range of observation is from H3 to 133 ®1* Per ®iaute. effective renal plasma flow are: Corresponding figures for the mean 266 ml. per minute, standard error 7 .6 , range 139 to U30 ml. per minute. A mean filtration fraction of O .317 in these dogs indicates that about 12$ of the plasma passing through the glomeruli of the kidney is filtered. 21 Tor experimental investigations certain anesthetic agents are required, to perform surgical operations in acute experiments or to facilitate pro­ cedures such as those required in the study of renal problems* Among the convenient anesthetic agents, sodium pentobarbital has been widely used* Mylon et al* (19^3) pointed out that certain side effects of the anesthesia may complicate the experimental conditions. The first systematic investiga­ tion of the influence of sodium pentobarbital on renal hemodynamics was made by Corcoran and Page (19^3)• was They reported that the renal function of dogs not impaired during a period of two hours1 anesthesia induced by 30 mg. per k g . body weight of sodium pentobarbital injected intraperitoneally. Sodium pentobarbital, 30 mg. per kg. given intravenously and additional small amounts to maintain uniform anesthesia for a period of five hours, has been found to induce a negligible effect on the sodium reabsorption mechanism (Selkurt and Glauser, 1951). Prolonged barbital or pentobarbital anesthesia of 5 to 6 hours* duration suitable for surgical purposes has no apparent effect on the glomerular filtration rate, although a decrease in the overall effective plasma flow and Tm for PAH may be observed (Glauser and Selkurt, 1952). In a study of renal problems, urine flow is of primary importance and the problem of diuresis is always encountered* Administration of water to rabbits induces marked increases in renal plasma flow, glomerular filtration rate and urine flow (Forster, I9H7). However, tinder controlled experimental conditions, a 52-fold variation in urine flow (0 .01^ - O .725 ml./kg./min.) has been found to have no effect on glomerular filtration rates in rabbits (Forster, 195*0. 22 It was Kirk (193^) who first reported a very low value for total amino acid clearance, ranging from 1 to S al, per minute in man. Doty (19^1) early found that a complete reabsorption of tyrosine and histidine took place in the dog when the filtration load was increased moderately, later, Pitts (I9U3 ) found that tubular reabsorption of glycine in dogs amounted to more than $8?o of the filtration load at normal plasma amino acid levels (below 10 mg. nitrogen per 100 ml.). The reabsorption of DL-alanine in the dog remains at a high level of 6*4 to Sl$ with high loads of *10 mg. nitrogen per 100 ml. (Goettsch et al., 19*4*4). Such essential amino acids as leucine, isoleucine, tryptophane, valine (Beyer et al., 19*46), threonine, phenylalanine (Busso et al., 19*47)* histidine and methionine (Wright et al., 19*47) 3X9 80 effectively reabsorbed by the tubules in the dog that their Tm values could not be reached with the dosages without causing severe effects on the subjects. In the human, onlya minutefraction of any excreted when subjects areplaced on a of theingested amino acids is diet containing 1 gm. ofprotein per kg. body weight per day (Harvey and Horwitt, 19*49; Kirsner et al., 19*49; Sheffner et al., I9US). HHepatlc Clearance”; lewis (19*48) first applied the concept of Mhepatic clearance” to the study of liver function in the similar manner that Clearance” has been applied to kidney function. He suggested the following formulas P - P' P x P' " C V where F is the initial plasma concentration, P' is the final concentration greater than zero, C is the volume of plasma cleared during this time inter­ val, V is the total volume of the fluid compartment containing the dissolved 23 material, and C/V is a fractional clearance which represents the fraction of the fluid volume (or solvent compartment) cleared during the clearance period. The utilization of this fractional clearance formula is based upon three assumptions: (1) relative constancy in fluid volume under different conditions, (2) removal of test substance is accomplished solely by the liver, and (3) the time-concentration is a simple logarithmic curve. With this technique, a clearance may be calculated from the changing plasma concentra­ tions. A more direct method for determining the clearance of the plasma by any organ or tissue is to compute the amount of the plasma which contains that amount of the test material accumulated or excreted by the organ or the tissue per unit time. Organ or tissue clearances of certain test materials have been studied for some years. The general theory has been summarized byStrajman and Pace (I95I). The term "disappearance clearance" indicates the rate at which ok a test substance is removed from the tissue. The removal of Ha from dif­ ferent tissues has been extensively studied in this connection. The term "accumulation clearance" indicates the rate of accumulation of a test sub­ stance in the tissue. The accumulation rate of iodine in the thyroid has been also extensively studied. In the present studies, the hepatic clearance 60 of Co is computed from direct measurement of the quantities recovered in the bile. 2k METABOLISM 0? COBALT 60 IN CHICKENS The purpose of the experiments with chickens is to compare the fraction of cobalt which is recoverable from the intestinal tract following oral administration with the fraction recovered following intravenous injection. Lata will be presented showing differences between recoveries from the various segments of the tract. An indirect estimate of the fecal loss and the urinary fraction in the bird will also be presented. A. Method Two mixed populations of birds culled from the poultry flock of Michi­ gan State College and averaging one kg. in body weight, were used. The necessary surgery was performed on the first day, and on the second day, 20 micrograms of Co^° solution* per kg. body weight was administered. Twenty-four hours later, the birds were killed and analyzed for radioactivity. Trial 1 consisted of 6*4- birds. The junction of the caeca and the large investine in each bird was pulled to the surface of the body through a ventro-lateral abdominal incision. In the first group of 16 birds, both caeca were left intact; in a second group of 16 birds, both caeca were ligated at the junction with the large intestine (see Introduction, p. 2); *Co^°, as Co&'SOty, obtained from Tracerlab, Boston, Mass. The stock solu­ tion containing HOO micrograms of cobalt per ml. of 0.1 N HC1 solution was diluted to about 70 micrograms per ml. (pH 2) with physiological saline. The 20 micrograms injected contained 10 microcuries of Co®0 . Physical decay corrections were made at each time of counting. 25 in a third group of 16 birds, the right caecum was ligated; and in the last group of 16 birds, the left caecum was similarly ligated. then closed with three or four stitches. Half of the birds in each group received the C o ^ injection through the wing vein. Co 60 The incision was In the other half, the was Injected directly into the lumen of the gizzard. After decapitation, the intestinal tract from the proventriculus down to the cloaca was removed, leaving behind the pancreas and mesenteries. Neither the cloaca nor the esophagus and crop were included. Each caecum was then removed and its contents mechanically separated from the caecal wall. The samples obtained were spread on the bottom of separate 20 ml. beakers and counted while moist above a thin mica end-window G-M tube. The standard, made by adding a known quantity of the Co^® injection solu­ tion to 2 ml. of water, was likewise counted in a 20 ml. beaker. The remainder of the intestinal tract was placed in a 250 ml. beaker and similarly counted. Due to the differences in the total volume of the intestinal tracts, a number of standards were prepared by adding a known quantity of to different volumes of water. The precise standard employed was that having the same volume as the sample of intestine being measured. Trial 2 consisted of 32 birds. The large intestine of each bird was ligated below the caecal opening into the intestine. In the first group of 16 birds, both caeca were left intact; and in a second group of 16 birds, the left caecum was ligated. The Incision was then sutured. Cobalt 60 was injected into the wing vein of half of the birds and into the lumen of the gizzard of the other half as in Trial 1. were killed. Twenty-four hours later, the birds 26 After dissection, the small Intestine and its standards were counted in a similar manner to that employed with the birds in Trial 1. The caecal wall, the caecal contents and the whole large intestine, however, were placed in separate porcelain crucible covers, and ashed at 500 to 600 degrees Centigrade in a muffle furnace for five hours. These samples were then counted under a thin mica end-window G-M tube. Standards for these samples were prepared in triplicate by pipetting a known quantity of Co solution onto the covers and drying. injection A self absorption correction was applied to each sample (Appendix 1). In both trials, one ml, blood samples were obtained in porcelain cru­ cibles by cutting the wing vein immediately before the killing. Two-tenths ml. of bladder bile was also taken from each bird and placed in separate porcelain covers. The total volume of bladder bile in each bird was recorded. All blood and bile samples were dried at room temperature for 2k to 36 hours and counted under a thin mica end-window G-M tube. Standards were prepared 60 by adding known quantities of Co to blood and bile taken from non-injected birds. All data were treated statistically. The groups1 means and their standard errors (Snedecor, 19^6) are summarized in the appropriate tables. Aberrant data were rejected (after initial statistical evaluation) according to the Chauvenet criterion (Calvin, 19^9) to give the population from which the final statistics were computed. B. Results The recoveries of Co^® from the digestive tract in both trials, computed as percentages of the injected dose, are summarized in Tables 1 to h and Figures 2 to 6 . 27 Trial It Large Intestine Not Ligated Intestinal Recovery. As shown in Table 1 and Figures 2 and 3, when the large intestine was not ligated, there were no significant differences in the Co go recoveries from the intestinal tract 2b hours after injection whether fin the Cow was administered intravenously or injected directly into the lumen of the gizzard. In the sham operation, the total recovery from the intestinal tract averaged 12,1$, out of which an average of 7*2$ was from the small and large intestines. The remaining *+.9$ was recovered from both the caeca. When both caeca were tied, the total recovery averaged only 7.6$» a- figure which is in close agreement with the 7*2$ recovered from the small and large intestines alone in the previous group. In this case, only a small amount of the injected dose (less than 1$) was recovered from both the caeca. With only one caecum tied, total recovery was between that recovered from the sham operated group and that recovered from the group with both caeca tied. However, due to large individual variations, there were no statistical dif­ ferences between any of these groups. Extracellular Fluid Recovery: The C o ^ recovery from the extracellular fluid was calculated from the whole blood concentration. It was assumed that the C o ^ enters the entire extracellular compartment and that it does not penetrate into the blood cells. Both assumptions will be demonstrated to be correct from the data of the dog experiments. crit value of If one assumes a hemato­ and 25$ of the body weight to be extracellular fluid in the chicken, recovery from the total extracellular space averaged 5.1+$ when all groups of birds were considered. Inspection of Table 1 reveals no signi­ ficant differences between groups injected intravenously or groups in which the Co^® was introduced directly into the lumen of the gizzard. 28 Caecal Recovery: A consideration of the recoveries from individual caeca showed that the ligation of one caecum does not affect the recovery from the caecum of the opposite side. Accordingly, Table 2 and Figure 4 were tabulated so that the Co^® recoveries from all the untied caeca may be com­ pared with recoveries from all the tied caeca. Comparisons may also be made between recoveries following intravenous or intragizzard injections and be­ tween recoveries from the left or right caeca. As seen from this table and figure, when the large intestine was not ligated, the Co recovery from a single caecum or its contents 2k hours after injection was independent of the route of Co 60 administration. No significant difference could be detected between right and left caeca. However, the recoveries were much less when the caecum was tied than when it was free. The recovery from a single caecum or from its contents averaged 2.3$ and 1 .8$, respectively, when the caecum was free; as compared with only 0.28$ and 0.03$, respectively, when it was tied. The Co^® recovery from the caecal wall was also independent of the route of administration and whether the caecum was on the right or left side of the bird. In addition, the presence or absence of a ligature did not affect the Co^® found in the caecal wall. The recovery from the caecal wall, averaged for all groups of birds under all conditions, was 0 .3*$. Trial II, Large Intestine Ligated Intestinal Recovery: As presented in Figure 5 a»d Table 3, when the large intestine was ligated at its junction with the small intestine, the total intestinal recovery (in the sham operation) 2k hours after injection was 25$ when Co^° was injected intravenously and 63$ when it was introduced directly into the lumen of the gizzard. The proportion of the recovery from the gut which was found in the small intestine was about 70$ when C o ^ was 29 injected intravenously and 90$ when it was introduced directly into the lumen of the gizzard. The recovery from hoth caeca averaged ^.5$ of the injected dose and that from large intestine 1.6$ of the injected dose regardless of the route of administration, Extracellular Fluid Recovery; The Co 60 recovery from the extracellular fluid was computed as before and found to average H.l$ in all groups of birds. This does not differ significantly from the value of 5***$ those groups of birds with large intestine not ligated. Caecal Recovery; It may be noted, in Table H and Figure 6, that when the large intestine was ligated, the Co 60 recovery from a single caecum and its contents 2k hours after injection tended to be higher when Co^® was injected intravenously than when it was introduced directly into the lumen of the gizzard. In the sham operation, the total recovery from a single caecum and the recovery from its contents averaged 2.6$ and 2 .3$, respectively, when injected intravenously as compared with 1 .9$ and 1 ,5$. respectively, when introduced directly into the gizzard. However, due to the large indi­ vidual variations, the differences between these recoveries were not sta­ tistically significant. Heither the presence or absence of a ligature, nor the route of administration, nor whether the caecum was on the left or right side of the bird significantly affected the Co^° recovery. The Co^° recovery from the caecal wall in this trial averaged 0.33$* a value no different from that found in the previous trial in which the large intestine was not ligated. Ratio to Blood: Table 5 and Figure 7 show the relative Co^® concentra­ tions in the caecal wall, and in the large intestine plus its contents relative to the whole blood level. The concentration ratio of caecal wall 30 to whole blood averaged 22 and of the large intestine plus its contents to whole blood averaged H9 in all groups of birds. There were no significant differences between any of these groups. Comparative Data from Both Trials: The ratios of the Co^® concentrations in the caecal wall to whole blood and in the bile to blood in both trials are presented in Tables 6 and 7 * and in Figures 8 and 9. 60 With the large intestine not ligated, the Co concentration ratio of caecal contents to whole blood in the untied caecum was 53 to 69 when Co was injected intravenously and 90 to 106 when it was introduced directly into the lumen of the gizzard (Table 6 and Figure 8). these ratios were 0.3 to In the tied caecum, When the large intestine was ligated, on the other hand, all concentration ratios were above 100 (10*1- to 1^7) with one exception. In that one case, the ratio was only 68 (when the caecum was tied and when C o ^ was introduced directly into the gizzard). The Co^Q concentration ratios of bladder bile to whole blood, as pre­ sented in Table 7 and Figure 9 , were less than 1 in those groups of birds with the large intestine not ligated and between 1.5 and 3*7 in which it was ligated. those groups Ratios in all groups receiving intravenous injection tended to be higher than those in the groups receiving intragizzard injection. However, these differences were not significant. As shown in Table S, there were no significant differences in the wet weights of either the caecal contents (group mean per caecum I.13 to 2.05 gm.), or the caecal wall (group mean per caecum 1.22 to 1.7*0 S®*)> or the large intestine plus its contents (group mean per bird 3.20 to 3.30 gm.), or in the total volume of the bladder bile (group mean 0.60 to 1.19 ml. per bird) between any of the different groups of birds in both trials. 31 a *i ■d S o % Ti rH *rj © P O ) P . © © 03 (V O© O *H »» 2 d P K m 8 ^ p O P * 4 C M ia tl -=f- * Jt • st o\ C M • rH * C M K3 • pH C M • rH 60 * H 60 • C M ti tl ti tl tl tl tl K3 • in 03 • rH » d■ as * in rH • Is- K3 • Jt in in • M D m Is- 60 o • pH C M C M C M C M i— • m C M A S +3 o n m © & •d C D0 o ® a o d *h H •rl P P to a o> 0) P H p d d < h 2 •a d «! p fO © o S P o p ► HP C ( © to • K3 11 in • C M rH • m • • • 03 » tl tl tl tl tl ti tl Is- O C M • r— O • o s0 « 60 03 • 60 M O • C M • O t d- O pH • pH pH • 60 rH Is- taOU a 0 © ttf) r— • 1 a • O H pH • H d o P © O « © p £ 2 o © © # s p © o © pq o m pH • C M tl • 3 &« • pH in » C M C M rH tl • tl tl ti tl tl tl =1• LTV 60 • m © 60 • • o m ♦ C M C M m • rH irv Is• C M 60 • C M Is• pH M 3 • rH r— • C M M3 • C M tl tl • m « S © pi m • o o d- o d 'd p •H «H M © © m p ^ -d o *pj © 12 nj tt 3 <5 © h rH a « 8 14 o 15 © 6fl b 8 “ © * * rH © *8 d *4 tl © C I* P p ss >» O to 84 E4 8 © «d O * *3 o rH M © Ph © © •h e © pH ►P * C M rl ti tl tl tl tl ti C M • 60 C M » M 3 pH • 03 Is« Is- r-• •d 8i © to N •H © P *H O o *H P O d « « O a SP P P d H p d PH o d1 04 M d © • o• ft >4 * M 3 in •d *d © h P © © © «P 4 o d © St u a b p d PH p d PH *4 3 © © •rl P © d © a S P d d P d PH P d PH in M 3 d U) © © •r4 T O*rl d o e I p o «d «P 4 © © © © •rl d o e Mean t standard •d © d Eh p *d 5 error b © >4 © rH «H 32 «H VO rH k 4» p 3 a 60 rH * o O tl tl tl rH o p o o « a ss o o » p ■3 O 0© © P 0 d o o o rH *1 tl tl ® 8 tl tl tl C M • o tl tl m Is* rH rH C M C M O o o o o 81 © in o « o o o $^ a Sc *H P rH © (6 © a © p0 eg iH C M H? § E h o © 0 M gs *H H ■3 o © © Be * 60 in 60 rH 1 — m in rrH 60 rH o O o o O* o tl tl ti tl • o tn m * o C M -rf • O f" C M • O ti o • o rN © © ft m Eh m rH OV o © o o tl ti tl Jt rH • o * o o a error 2 ap IT\ VO n iean t standard 6.0 O rH % § a © p * p p © o •fj © a •fj> H 0 H 6h O 0 © p P $ © «H o © n Pi ft s© «H P4 £* B* © > d d •-3p S to o d « H © fi q> «h gap as * $ 1^* m vo cvj’ rH o • tl ti tl tl H in -d* » 6 ♦ H CVJ o ♦i tl tl ti m VO cr. CVJ in m • C V J * dt 1 •d I ■p a ti o *h fi d * © d rH «rl •o 3 » a P© TO vo GO O vo ti tl cr> VO• jo­ a in «d •d d © <0 d O T ) a <0 Ip © C D C O © a -h id O Eh 3* TABLE k 60 Co Becoveries (Per Cent of Injected Dose) from Caecal Wall and Caecal Contents Twenty-four Honrs After Injection. The large intestine was tied off at its junction with the small intestine. Treatment Boute of Injection Caecum Caecal Wall Caecal Contents Total Bight 0 .3S 2 0 .20* 2.23 ♦ 1.13 2.61 2 1.50 Left 0.30 ± 0.07 2.30 2 0.30 2.60 2 1.3^ Bight 0.1*3 * 0.18 1.1*1 ± 0.71 l.SU 4 0.80 Left O .38 2 °*27 1.66 v 1.33 2.0l* 4 I.56 Bight 0.27 * 0.11 2.00 4 1.1*1 2.27 4 1.51 Left 0.25 ± 0.10 2.12 * 0 .9S 2.37 2 1.03 Bight 0 .3!* ± 0.17 1.01 2 0 .3I* 1.35 2 0 .1*1 Left 0.26 ♦ 0.10 O .69 2 0.22 0.95 i 0.27 Intravenous Sham Intragizzard Intravenous Left Caecum Tied Intragizzard * Mean * standard error 35 TABLE 5 C o ^ Concentration Ratios of Caecal Wall and Large Intestine plus Its Contents to Whole Blood* Twenty-four Hours After Co Injec­ tion. The large intestine was tied off at Its junction with the small intestine. Treatment Route of Injection Concentration Ratio Caecal Wall Large Intestine plus Ita Contents Blood Blood Left Right Intravenous 19 21 58 Intragizzard 23 26 kk Intravenous 19 IS kk- Intragizzard 2k 26 51 21 23 U9 Sham Left Caecum Tied Mean * Ratios to plasma would be 0.6 of the recorded values if a hematocrit of **0$ le assumed and no cobalt enters the cells. 36 TABLE 6 60 Co Concentration Ratio of Caecal Contents to Whole Blood* Twenty'four Honrs After Co Injection Routes of Injection Treatment Intravenous Concentration Ratio Caecal Contents; Blood Left Caecum Right Caecum 69 60 103 106 Sham Intragizzard Large Intestine Intact Both Caeca Tied Intravenous 3.3 3.0 Intragizzard 0.3 0.5 Right Caecum Tied Intravenous Left Caecum Tied Intravenous 2.h 69 Intragizzard 2.0 90 Intragizzard 53 1.8 101 0.9 Intravenous 119 117 Intragizzard 136 1*7 Intravenous 120 10k Sham Large Intestine Tied Left Caecum Tied Intragizzard 62 127 * Ratios to plasma would he 0.6 of the recorded values if a hematocrit of is assumed and no cohalt enters the cells. 37 TABLE 7 6o Co Concentration Ratio of Bile to Whole Blood* Twenty-four Hours After Co®® Injection Treatment Route of Injection Concentration Ratio Bile: Blood Intravenous 0.92 Intragizzard 0.2S Intravenous 0 .5S Intragizzard 0 *1^ Right Caecum Tied Intravenous 0.19 Intragizzard 0.15 Left Caecum Tied Intravenous O.63 Intragizzard 0.32 Intravenous 3.7 Intragizzard 2.3 Intravenous 3.* Intragizzard 1.5 Sham Both Caeca Tied Large Intestine Intact Sham Large Intestine Tied Left Caecum Tied ' * Ratios to plasma would he 0,6 of the recorded values if a hematocrit of is assumed and no cobalt enters the cells. 38 © © tt ©< rl P MP H L n 3 © a a P fl m p ►3 -*3 5* fl ©HO *H P . O n VO CTv * o o O ov « o ♦I tl tl tl K\ C V J CVJ CVJ KV K V kv kv Jfr C V J kv vo o o o • • p * © si O I rl Ui ♦I r~* © Et • tl xt • ti tl kv in • © •H 3 s ■ cd tt Q > P A iH iH kv vo kv o o t ©be 6= O P © p < H © •4 o ♦I vo rl « tl ti tl in C V J C V J 4 •d • p .© 3 CO n • 4 H 3 ►4 in 4 o o o H H r~« 60 CO o o o tl ti ti ov 60 r~- tl tl C O o tl O N xt O o ti tl r- kv a i co 6h ”3 a O © ©P © © o o o ° .i a P S I si ■ » * o tl C V J x t o tl p © *4 Jt IT\ o © o o tl tl h- L T v tl o tl xt CO VO k- o o o o tl tl ti in in C O o tl 1 * “ • o vo o * o tt o C V J o C V J rH C V J Xt in o o KV 4 C V J « tl K V tl CVJ tl tl r- I— H 6s 1 §1 VO « tl ♦ o tl © p M si + > s i rH A © o o o 8 o © © «tJ © ® >d H g * © *4 © O H fq H t) m O *4 o p .© CO I* P & * si *4 O 0 «H P © o p © 1 fl f© t-i ir\ u rv KV o o o ti tl CVJ C V J © tl 3 5, © © © © © P p p © o o tl •h r~- VO o o 3 (9 N N kv C V J •d •d « © o o kv © P tt 1 © © © © M p s p ■ w •d © © © N © p © o « # 1 tl vo CTv VO4 tl o o l> p © © •rl o tt S © © © © P to rJO B xt o *d p © © o ©a n Wo B P © © a « © ©h p © £4 © © P P P © © © C tO© » d In p © © ©H hi H B © O ■d © © o *d « H© © p o © © tt O CO © © ©p p ©© © hi H H © © © © © © p KV • o tl vo • o •d § o © m P o •d 3 tt N « © © p P p 43 O «d CO © © p © at WOB «5 © © © © p o -d « ©© » © d s* ©oii 39 TABLE 9 Metabolic Balance of Co^O Following Intravenous and Intragizzard Injections* All data are presented as percentage of injected dose* Intravenous Large Large Intestine Intestine Ligated not Ligated Injected Recovered from Whole Intestinal Tract 100 100 100 100 12 25 12 63 From Small Intestine From Large Intestine From Both Caeca Recovered from Extra­ cellular Fluid Intragizzard Large Large Intestine Intestine not Ligated Ligated 7 5 5 IS 2 5 *.6 7 5 5S 1.3 3.6 5 k Fecal Loss (by calculation) 13 0 51 0 Urinary Loss and Tissue Retention (By calculation) JO JO 32 33 Uo 123 INTRAVENOUS INJECTION 12 □ INTRAGIZZARD INJECTION / 2 3 4 CAECUM UNTIED BOTH CAECA TIED RIGHT CAECUM TIED LEFT CAECUM TIED 8 ✓ / X X ✓ / / / / / ✓ ✓ / / / / / / / / /, / 2 INTESTINAL 4 TRACT 1 2 ___ s / / / / / / / / / / / / 3 4 EXTRACELLULAR FLUID Figure 2. Co 6 0 Recoveries (Per cent of Injected Dose) from Intestinal Tract and Extracellular Fluid Twenty-four Hours After Injection. The large intestine was not ligated. The percentage recovery in extra­ cellular fluid was computed from whole blood activity as indicated in the text. Ul § s O INTRAVENOUS INJECTION □ INTRAGIZZARD INJECTION s / CAECUM UNTIED § 2 BOTH CAECA T/ED 3 RIGHT CAECUM TIED 4 LEFT CAECUM TIED <3 kl ! INTRAVENOUS INJECTION INTRAGIZZARD INJECTION CAECUM UNTIED BOTH CAECA TIED RIGHT CAECUM TIED LEFT CAECUM TIED LEFT CAECUM RIGHT CAECUM _EL L R L R L R L R L R L R L R L R L R L R L R L R I LARGE INTESTINE INTACT 4 LARGE INTESTINE TIED Figure 8. Relative Co^o Concentrations in the Caecal Contents to Whole Blood Twenty-four Hours After Co60 Injection. RELATIVE 0 CONCENTRATION Co6 ^7 i 0 INTRAVENOUS INJECTION □ INTRAGIZZARD INJECTION /. CAECUM UNTIED 2. BOTH CAECA TIED 3. RIGHT CAECUM TIED § 4. LEFT CAECUM TIED § o IF F71 ii ii ii ii * * 4 n 1 2 n El n 3 4 LARGE INTESTINE INTACT . n / 4 LARGE INTESTINE TIED Figure 9 Relative Go60 Concentrations in the Bile to Whole Blood Twenty-four Honrs After Co60 Injection. ks C. Discussion When the large Intestine was not ligated, recovery of G o ^ from the intestinal tract or its appearance in the extracellular fluid 2k hours after injection is not affected by routes of administration used in these experi­ ments (Table 1 and Figure 2). These results would he anticipated if the organism approached an equilibrium in which C o ^ is partitioned in a uniform and standard manner between body fluids, tissues and intestinal contents. Evidence for such a standard partitioning may also be drawn from the data 6o obtained by Monroe e£ al. (1952a) within 2k hours after intraperitoneal Go injection. Co Thus, it appears that the time scale for exchange with the various compounds of the bird is such that "uniform labeling" is attained in about a day's time. The route of administration is of no consequence for events after 2k hours. This is in contrast to the findings in the ruminants summarized by Comar (19^8) and reaffirmed in a subsequent report by Monroe et al. (1952b)* In the ruminants, the time required to reach the "uniform labeling", in the above sense, was much longer than 2h hours. Since the ruminants have an extremely large volume of bacterial flora in the rumen, a large amount of the orally administered C o ^ might be absorbed by the bacteria rather than by their host* 60 Consequently, the tissue content of Co following oral administration would be expected to be less than that following intravenous injection. In addition, the rate of flow of materials through the intestinal tract of the ruminants is so slow that considerable time must necessarily elapse before an appreciable fraction of C o ^ in the tract can leave the animal. Since the transport rate of materials through the intestinal tract is slow, 1*9 it might he thought that there would he a greater opportunity for the absorp­ tion of the Co^° in ruminants. The fact that low absorption was found in sheep following oral administration (Monroe et al., 1952b) is a direct evi60 dence for large scale Co binding by the intestinal contents in this species. 60 These data also indicate that Co passes across the intestinal wall in both directions. The intestinal absorption of inorganic cobalt has been demonstrated by separate experiments in this laboratory. WoIterink and lee (unpublished data) found that 50$ of the administered dose is absorbed within 20 minutes in day-old baby chicks when Co^°, as Co^SOij., is injected directly into the lumen of the gizzard. Fandurang (1952) has also been able to show 60 that inorganic Co is absorbed from the tied caecum of the chicken. The 60 intestinal "excretion” of Co will be discussed later. At the end of 2^ hours, a total recovery from the entire intestinal tract of 12$ when the large intestine was not ligated (Table 1 and Figure 2) and of 25$ when it was ligated (Table 3 s M Figure 5) was found following intravenous injection. At least .13$ of an intravenously injected dose, then, passes out through the feces during the first day. Out of the 75$ covered from the intestinal tract of these birds, only 5$ extracellular fluid. re­ found in the A large fraction of the remaining 70$ must have been excreted in the urine. On the other hand, in the case of intragizzard injection, 12$ of the injected dose was recovered in the entire unllgated intestinal tract at the end of 2b hours, whereas 63$ was recovered if the large intestine was ligated. Thus, at least 51$ of the dose administered into the gizzard appears to be lost in the feces during the first day. In addition to the U to 5$ found in the extracellular fluid, a large fraction (up to 32 to 33$ 50 of the administered dose) of the 37$ aot recovered in the intestinal tract must have been excreted in the urine. In the dog experiments, 35 to 60$ of an intravenously injected dose was eliminated in the urine during the first 7 to 13 hours (Table 19). urine is the chief pathway for the excretion of Co Thus, in both the dog and the chicken. A summary of the metabolism balance of Co followin g these two routes of administration is tabulated in Table 9 , C(\ In this table, the Co recovery from the extracellular fluid was com­ puted on the assumptions of an average hematocrit value of cellular fluid value of 25$ of the body weight. radioactive Cl 38 Ho$ and an extra- By means of thiocyanate, 2h 22 , Ha , or Na , the extracellular space has been observed to be from 21 to 23$ of the body weight in the rabbit (Hahn and Hevesy, 19^1; Manery and Eaege, 19^1), dog (Greenbert et al., 19^3a » tinkler et al., I9H3), rat (Cuthbertson and Greenberg, I9H5)* and in the adult human being (tellers et al., I9H9 ; Kaltreider et al., I9HI). A higher average value of Hi to HH$ of the body weight was found for infante (Fellers et al., I9H7). I9H9; Flexner et al., For normal chicks (13 to 35 days of age with a body weight ranging 121 to Hll gm.), a thiocyanate space of H3$ of the body weight and a hemato­ crit value of 30.5$ have been reported (Hegsted et al.» 195^)• Although values for older chickens are not available, in view of the data cited from other species, it seems reasonable to use a value of 25$ of the body weight for extracellular fluid and of that the Co Ho$ for the hematocrit. It should be emphasised recoveries from the extracellular fluid were statistically the same in all groups of birds and in both trials. This means that Co gn retained in the body fluids reached a constant level in 2H hours whatever the route of administration and whatever the route of passage out of the body. 51 After intravenous injections, an unexpectedly large fraction of the Co fin was recovered from the intestinal tract. In the dog experiments (Table 19), hepatic bile was found to carry from 5 to 10$ of the total intravenously injected dose in periods of 7 to 13 hours. Significant amounts of cobalt have not been eliminated in the pancreatic juice (Sheline et al., I9H5-I9H6). fin This suggests that Couu is "excreted*1 into the small intestine through the bile. 60 However, in chickens the low Co concentration in bile and the low bile to whole blood concentration ratio indicates that an appreciable fraction 60 of the intestinal Co recovery may reach the small intestinal lumen directly through the wall. When the large intestine was not ligated, only a very small amount of fin Go u was found in the contents of the tied caecum regardless of the route of administration (Table 2 and Figure H). However, recoveries from the caecal contents in the untied caeca were more than 20 times as high. fin from the gut. that the caecum receives most of its Co actually enters the caecum across the caecal wall. This indicates Only a small fraction This is demonstrated further by the fact that the C o ^ concentration ratios of the caecal contents to whole blood were below H in all the tied caeca and above 50 in all the untied caeca (Table 6 and Figure 8). When the large intestine was ligated, on the other hand, the ligation of 60 caecum did not cause any significant decrease in the caecal Co recovery (Table H and Figure 6). 60 This implies that large amounts of Co can enter the caecum across its wall under appropriate conditions. These conditions are evidently satisfied when both the large intestine and the caecum are ligated. fin passage across the caecal wall is due to an altera­ Whether this large scale Co tion of the caecal function caused by ligation or is due merely to a large 52 reservoir of Co 6o maintained in the body for a longer time requires further investigation. Little information concerning the blood and lymphatic supplies of the caecum in birds is available. The ligation of the caecum was made around its neck and close to the junction with the intestine. Surgical damage to the mesentery around the caecum was avoided as much as possible. The place* ment of a ligature appeared to have little effect on the blood and lymphatic circulation of the caecum, judging from the observation that Co recoveries from the caecal wall were about the same in all groups of birds and in both trials (Tables 2, U, Figures k and 6). Thus, there is little evidence for large scale circulatory impairment in the ligated caeca. In view of the small mass of the large intestine plus its contents (group mean, 3.20 to 3*3° S®» wet weight, Table 8), the 1 to 2$ C o ^ recovery from the large intestine when the latter was ligated (Table 3 s&A Figure 3) i® very significant. The high Co concentration ratio of ^9 for the large intestine plus its contents to blood (Table 3 &hd Figure 7) suggests that the /Ta Co is actively secreted into the large intestine in the bird. Cobalt 60 might have entered the large intestine by aatiperistalsis from the cloaca into which the ureters empty. Although no estimate can be made of the fraction of the continuous flowing urine which is forced back into the large intestine, it seems unlikely that any large amount of the Co^° recovered from the large intestine could have come from this source. The existence of high C o ^ concentration ratios in the wall and/or in the contents of the intestinal tract to whole blood strongly indicates that 53 the 6o must Co he in aome ,rbound" form or forms*. B12 containing Cow The presence of vitamin has recently been reported both in the excreta and in tissues including small intestine (Davis and Chow, 1951; Monroe et al., 1952b). In addition, Monroe et slU (1952b) have found what they designated as •‘bound11 Co^° in the excreta and ia tissues of sheep. Pandurang (I952) of this laboratory has also claimed evidence for the presence of some unknown bound form of C o ^ in the caecal contents as early as one hour after the 60 inorganic Co was injected into the lumen of the tied caecum in the chicken. 60 Apparently, a large amount of the Co is present in tissues and in intes­ tinal contents in some “bound" form or forms. The highest Co concentration ratios were found in the caecal contents, 60 Indicating that the "non-diffusible" Co was present in larger quantity in the caecal contents than in the caecal wall. This implies that either the size or the metabolic activity of the intestinal bacterial population may be fin directly concerned with the "binding" of Co • Davis and Chow (195^) have reported that the fecal concentration of vitamin 60 containing Co activity can be increased by the incorporation of aureomycin in the diet or by fin increasing the level of inorganic Co in the ration of rats. They attri­ bute the effect of the antibiotic to some alteration in the intestinal bacterial flora. The existence of "non-diffusible" Co 60 in the intestinal wall as well as in the contents may be the result of "continuous diffusion" of inorganic Co**® from the circulation through the wall into the lumen of the intestinal tract. In passing through the intestinal wall, and again after entering the 60 lumen, some fraction of the Co may be complexed into large "non-diffusible" *The term "bound" is used to indicate a form of cobalt which does not leave the cell despite a concentration gradient favoring diffusion out. These "bound" form or forms of Co®® may be true chemical compounds; or they may be coordi­ nation complexes; or they may be simple adsorption complexes. 5* molecules. The "diffusion* process might continue until the wall and the contents of the intestinal tract can no longer hind the Co^® into its "nondiffusible" form or forms and a "saturation" point reached. 60 As shown in Table 6 and Figure 8, the Co concentration in the caecal contents varies under different experimental conditions. These concentra­ tions were higher following intragizzard injection than following intravenous injection, and they were highest in those groups of birds with the large intestine ligated. 60 These differences in Co concentrations in the caecal contents may be due to one or more of the following reasons: 1. The total integrated Co^® in the body fluid available for "diffusion" into the caecum during the 2*4 hours of the experiment might be higher under one set of experimental conditions than under another. 2. The number of bacteria in the caecal contents might vary under different experimental conditions in spite of the fact that the wet weights of the caecal contents are about the same (Table 8). 60 The uptake of Co per bacterial cell might also vary under different experimental conditions. 3. Certain caecal functions, for example the ability of the caecum to absorb water, might be altered by the presence of ligatures, despite the fact that ligation causes no apparent effect on the blood and lymphatic circulation of the caecum. If water transport is interfered with by mechanical stimulus, it would be reasonable to expect alterations as well in the transport of water soluble materials containing Co&>. Monroe et al. (1952a) found a very low concentration of Co^O in the - 60 liver of young chicks after intraperitoneal injection of inorganic Co . In the present experiments, the concentration of Coou in the bladder bile of the chicken 2k hours after oral or intravenous administration was much lower than that found in the hepatic bile of the dog. The Cow concentration ratios of the bladder bile to whole blood of the chicken were below 4 in all groups of birds. No information can be found with respect to the quantity of bile flow or to the emptying time of gall bladder in the bird. However, in 2k hour8 , emptying of gall bladder must certainly have taken place. This 60 * eliminates the possibility that the Co -containing 24-hour hepatic bile was diluted by Co^®-free bladder bile formed earlier. Since hepatic bile is concentrated within the gall bladder by the removal of water, the concentra­ tion ratio in the hepatic bile should be still lower than that observed in the bladder bile. In view both of this low concentration ratio and of the low volume of bladder bile, the liver of chicken apparently does not remove cobalt from the blood to the same extent as does the liver of the dog, Further investigation concerning the function of the liver in the excretion fin of Co in the chicken is needed. 56 METABOLISM OP COBALT 60 IN DOGS The purposes of the experiments with dogs are: to determine rates for hoth Biliary and urinary excretion of coBalt, to evaluate the function of the renal tuhules in handling this trace element and to tionof cohalt Between the Body fluids. determine the parti­ Comparisons will Be made Between the Biological turnover rates for the removal of inorganic coBalt and amino acid coBalt complexes from the peritoneal cavity and the Blood. Data will also Be presented showing the relative distributions of coBalt in the intestines and tissues following different routes of administration. The reaBsorBaBility of the coBalt excreted in the Bile and urine collected from the experimental dog will Be studied By determining their intestinal absorp­ tion in chicks. A. Methods General Remarks: Twenty-one mongrel dogs, weighing Between 5.5 and 21.B kg., and 850 three-day-old White Leghorn chicks were used in these experiments. The C o ^ used for all injections was the same diluted solution (about 70 micrograms per ml. with a pH 2) used in the chicken experiments. Infusion solutions and an injection solution of second dilution (0.813 microgram per ml.) were prepared from the first diluted solution in physiological saline. All Blood, urine, Bile and plasma samples used for radioactivity deter­ mination were dried in separate porcelain crucibles at room temperature for 2k to 36 hours. Standards were prepared from known quantities of Cow added to Blood, urine, Bile and plasma collected Before injection of Co 60 . 57 All standards were made 1st triplicate. each dog. Blood standards were preoared for The other standards were used throughout these experiments. All the plasma and urine samples were refrigerated "before chemical analysis. The group means and their standard errors were computed as in the chicken experiments. Surface area was calculated from the Meeh-Hubner weight formula: S.A. = 0.667 11.2 x Wt. 100 where S. A. is the surface area in square meters and ft. is the body weight in kg. Cr\ Distribution of Co in Blood: Thirty-four blood samples were collected from the jugular vein of Dogs No. 15*, 16, 21, Zk and 25 at various intervals after intravenous 60 injection of inorganic Co . Radioactivity measurements from each sample were made on one ml. of heparinized whole blood, on one ml. of plasma and on 3 ml. of the cadmium hydroxide plasma filtrate which was used for the deter­ minations of creatinine and PAH. A standard for the plasma filtrate was 60 prepared by adding a known quantity of Co to water. The plasma filtrate samples and the standards, in triplicate, were dried in separate porcelain crucibles at room temperature for 96 hours. The hematocrit determination on each blood sample was made by spinning the well-mixed heparinized whole blood in a fintrobe hematocrit tube for half an hour at 2250 RPM within one hour after sampling. A change of the cell ♦Dog No. 15 was an unsatisfactory preparation for the study of renal clearance of Co . However, two blood samples were drawn 1 and 2 hours after the intravenous injection of Co • 58 volume "by less than if was observed after an additional half hour of centri­ fugation at the same speed, indicating satisfactory packing of the red blood cells. A correction for the plasma trapped between cells was made using a factor of 0 ,9 6 (Gregerson and Schiro, I938). Between the time of sampling and centrifugation, blood samples were refrigerated. From the above pro­ cedures, the partitioning of C o ^ between cells, plasma and protein-free plasma filtrate was computed. 60 One dog (So. 26) received 20 micrograms of Co per kg. body weight intravenously. Two blood samples of about ^K) ml. each were drawn from the jugular vein at one and ten hours after injection and heparinized. After centrifugation 10 ml. of plasma was pipetted into a dialyzing tubing* about four inches in length. The protein in another 2 ml. of plasma was precipi­ tated with cadmium hydroxide as described in Appendix 2. Ten ml. of the supernatant protein-free filtrate was pipetted into a second dialyzing tubing. After all the plasma filtrate was decanted, 20 ml. of distilled water was added to the protein precipitate. this precipitate. tubing. Vigorous shaking yielded a suspension of Ten ml, of the suspension was put in a third dialyzing All tubes were dialyzed against running tap water for eight to thirty hours. Frequent shaking of the tubing containing plasma or protein precipitate was necessary to prevent packing of the denatured proteins. Radioactivity measurements were made on one ml. of plasma, on 3 ml* of protein-free plasma filtrate and on 3 ^1* ot precipitate suspension both before and after dialysis. All samples were prepared in triplicate ♦Dialyzing tubing, Chicago Apparatus Company, Chicago, Illinois 59 and dried for k8 to 72 hours. The percentage of Co^° diffusible through the cellophane membrane was then calculated. 60 Rate of Co Absorption from the Peritoneal Cavity and Its Bate of Elimina­ tion from Blood When Injected Intraperitoneally; fiO Twenty micrograms of Co per Isg. body weight was injected intraperi­ toneally into each of four dogs weighing between 7 and 10.5 kg. received the Co^® solution. cysteine. Two dogs The other two received a complex of C o ^ with To make the Co^-cysteine complex, the pH of C o ^ solution was first adjusted to between 6 and 7* A sufficient amount of cysteine* was 60 added at room temperature to give a Co /cysteine molar concentration ratio of 1:30. This mixture contains ten times as much cysteine as is required for 60 the formation of the Co -cysteine complex (Michaelis and Yamaguchi, I929). About 2 ml. of blood were drawn from the cephalic vein at intervals of 5, 10, 15, 3 0 , 60 minutes, and 2 , 3 , h, 5 , 6 , 7 t 8 and 20 hours after injection. Radioactivity was measured on one ml. of whole blood. Disappear­ ance curves from the peritoneal cavity and blood were then plotted on semilog paper following the method of Berlin and Siri (195b). Reabsorption of Co Cr\ from the Urine and Bile Samples: The hepatic bile and urine samples from Dog No. 7 were used. Pooled samples collected between 0-U-, 4-S, and 8-12 hours after the initial Cow injection were employed. The pooled samples were then diluted with physio­ logical saline to the concentration indicated ia Table 12 and kept in the refrigerator overnight. Seven groups of three-day old White Leghorn chicks were set up, each group consisting of eleven birds. Three groups of these Bine hydrochloride. C. P., Pisher Scientific Co., Pittsburgh, Pennsylvania. 60 chicks received a urine injection; three groups, a hile injection; and one £T/*\ group, an injection of an inorganic Co u solution. One ml. of these samples fin or of the Cow solution was injected directly into the lumen of the gizzard of each chick. All chicks were killed half an hour later. The intestinal tract from above the proventriculus down to the cloaca was removed and placed on individual tared procelain crucible covers. They were then ashed in a muffle furnace at 500 to 600 degrees Centigrade for five hours, weighed, and counted under a thin mica end-window G—M tube. adding known quantities of Co 60 Standards were prepared by solution to the intestinal tract removed from non-injeeted chicks and ashed and counted in the same manner. Since the weights of all the ashed samples were about the same (group means, 9.6 to 10.5 mg./cm2), no self-absorption correction was applied. These urine and bile samples and, in addition, those from Dog Ho. 5 (which received the same treatment as Dog Ho. 7) were also studied by means of paper partition chromatography and radioautography. All samples were spotted on Whatman Ho, 1 filter paper and developed by ascending technique (Williams and Kirby, 19*4-8) with n-butanol saturated with water for a period of 72 hours at room temperature. Radioautograms were prepared by placing Kodak Ho Screen X-ray film in direct contact with the resultant paper chroma­ tograms for a period of J2 hours, 60 Renal Clearance, Tabular Reabsorption and Hepatic Clearance of C o . A total of fourteen dogs, weighing 5.5 to 21.** kg., were employed in this section of the experiment. JPood was taken away from each dog for about ten hours prior to the experiment. Half an hour before each experiment, 50 ml. of water per kg. was given by stomach tube. Each dog was anesthetized with 30 mg. of sodium pentobarbital per kg. body weight. In those experiments of 61 long duration (up to 13 hours), additional doses of 3 to 5 mg. of pento­ barbital per kg. were administered as necessary to maintain deep slow respiration. Six of these fourteen dogs’1' received an intravenous priming dose of 50 mg. of creatinine** and of 2 mg. of PAH (sodium p-amino hippurate)*** per kg. half an hour prior to the beginning of the clearance periods. At that time, infusion of solution containing 0.2$ of creatinine and 0.1$ of PAH was begun through the exposed femoral vein at the rate of 200 ml. per hour using a mercury drop method. The urine samples were collected from the bladder of four dogs through an indwelling catheter and by supra-pubic cannulation of both ureters with plastic tubing in the remaining two dogs. In cases where the bladder urine was collected, each period was terminated by a single rinse with a known quantity of physiological saline and an air-wash. Three or eight consecutive urine collection periods of 15 minutes' duration were run in each of the six dogs beginning half an hour after a single intra60 venous injection of 20 micrograms of Co per kg. Blood was drawn from the jugular vein opposite to the infusion side and heparinized. In four other dogs, the cystic duct was ligated and the common bile duct cannulated with 1 mm. I.D. plastic tubing through a ventral abdominal incision. Bach ureter was similarly cannulated. The trachea was cannulated with a metal T-cannula and the femoral vein with a glass cannula connected *0ne of these six dogs, Dog No. 1 5 , was an unsatisfactory preparation. She died one hour after a single intravenous injection of 20 micrograms of Co per kg. body weight. •♦Creatinine, C. P., Pfanstiehl Chemical Co., Waukegan, Illinois. ***PAH, Sodium para-aminohippurate, 20$ solution, Sharp and Dohme, Philadelphia, Pennsylvania. 62 to an injection burette. Arterial M o o d was sampled as follows: An 18-gauge needle with its adapter end removed was connected to a 1 mm. I. D. plastic tubing, with a close-fitting wire serving as a ping. The needle was passed obliquely through the wall of the artery (the carotid, except in Dog No. 3 , which was sampled from the femoral). After sampling, the wire plug was dipped in heparin and inserted through the plastic tubing to the tip of the needle, which was left in situ. An artery clip was placed on the tubing to prevent leakage. Occasionally a soft clot formed in the needle. This was easily pressed out by clamping both ends of the artery and applying pressure in between. About 2 ml. of blood were taken at each sampling and heparinized. Bach of these four dogs received 20 micrograms of Co^® per kg. body weight from the injecting burette, followed by 5 ®1. of saline immediately before the urine and bile collections. In the remaining four dogs, similar operations were made to collect urine and hepatic bile through ureteral and biliary cannulae. The trachea Two of these four dogs (Dogs No. 6 and 7) received a go priming intravenous injection of 10 micrograms of Co per kg. followed by was also cannulated. a constant infusion of a solution containing, respectively, 37.56 or 25.23 60 micrograms percent of Co in physiological saline. Infusion was made through an exposed femoral vein at the rate of 100 ml. per hour using the mercury drop method. Urine and bile collections were made as in the four dogs 60 which received only a single injection of Co . In the last two dogs (Dogs No. 2h and 25), an intravenous priming dose of 10 micrograms of Co 60 50 mg, of creatinine and 2 mg. of PAH per kg. body weight was given half an hour prior to the beginning of the clearance observation. The infusion solution consisted of 26.57 or 23.38 micrograms percent of Co^°, 0.3# of , 63 creatinine and 0.15$ of PAH in physiological saline. The Infusion rate was for the same as the two previous dogs. Clearance observations were made at three different stages during the experiment. Each stage consisted of three consecutive urine collection periods of 30 minutes* duration* The first stage was started half an hour after the initial Co^° injection; the second stage, six hours; and the third, ten and one-half hours. Bile samples were also collected during the urine collection periods. Badioactivity measurements in all these fourteen dogs were made on one ml. of whole blood, on 0.05 to 0 ,2 ml. of diluted urine, on 0.1 to 0 .2 ml. of the hepatic bile and also, in some cases, on one ml. of plasma. Creatinine clearances were used to measure the glomerular filtration rates and PAH clearances to measure the effective renal plasma flows. Creatinine was determined on the cadmium hydroxide plasma filtrates (Pujita and Iwatake, 1931) and on diluted urine by the alkaline picrate method (Folin and Wu, 1919)* (19^5). PAH was determined by the method of Smith et al. All analyses were made in duplicates. The detail methods of these chemical analyses may be found in Appendix 2. A plasma concentration value interpolated to coincide with the midpoint of the urine collection period was used to calculate the clearances, Tm for PAH and Co load during that period. Bile samples were collected in 15 ml. graduated centrifuge tubes and urine in 10, 50 or 100 ml. volumetric flasks depending upon the length of the collection period and the urine flow. samples were capped to prevent evaporation. All To determine the urine volume, each urine collection flask was filled to the mark with distilled water from a 50 ml. burette. The difference between the total volume of the flask and water or saline and water added gave the urine volume. One ml. of each of 6U the urine samples diluted as described above were again diluted to 100 or 250 ml. in another volumetric flask. Determinations of creatinine and PAH were made on these final dilutions. Data derived from the above determination were computed by the use of the formulae listed in Appendix 2. These include renal clearance of creatinine in ml. per minute (Ccr), renal clearance of PAH in ml. per minute (0pATy), renal clearance of Co 60 in ml. per minute (Oq06o) ♦ tubular secretory mass of PAH in mg. per minute (Tm), filtration fraction (PP), C o ^ load in micrograms per minute, C o ^ excreted in micrograms per minute, C o ^ reabsorbed in micrograms per minute, and 0o^° reabsorption in percentage. Tissue Distribution of O o ^ in Dogs: Two dogs of 10.0 and 11.0 kg. were anesthetized with 30 pentobarbital per kg. body weight. sodium Through a ventro-medial abdominal incision, three small intestinal loops, each four inches in length, were ligated at both ends. The first loop was at the first part of the duodenum with the bile duct emptying into its lumen. The second loop was at the last part of the duodenum and the third loop, at the beginning of the jejunum. received 20 micrograms of Co^° per kg. intravenously. One dog The same amount of Co^° was injected into the lumen of the second intestinal loop of the second dog. With the abdominal incision sutured up, these two dogs were placed back in the cage and supplied with water only. 60 Twelve hours after the injection of Co , these two dogs were anesthetized again. Small pieces of the liver, the spleen, the right kidney, the three loops of the small intestine, the caecum and the first part of the large intestine were then removed. All segments of the intestinal wall, including caecum, were separated from their contents. Each sample, about 0.5 to 2.0 gm., 65 was wet-weighed, ashed, ash-weighed and counted in separate porcelain crucible covers. The second loop of small intestine from the dog which received intra- intestinal injection of Co was digested with a minimum amount of concentrated nitric acid until all solid matter had been dissolved. then boiled off. The excess acid was The solution was cooled and diluted with acetone and a little water to form a clear solution of 250 ml. Triplicate samples, con­ taining 2 ml. of) each of this diluted solution were placed on separate porcelain crucible covers, and dried, ashed, ash-weighed and counted in a similar manner. Standards were prepared in triplicate by adding a known quantity of C o ^ to the crucible covers. A self absorption correction (Appendix 1) was applied to all these samples. Radioactivity measurements on one ml. of blood, plasma, bladder urine and bladder bile of these two dogs were also made. The various treatments of all these twenty-one dogs are summarized in Table 10. •d o I o *H «H Pi P +» © 0 -rl *H o o © o © «H .0 o j3 o +» -p p 0 +» O © «H s B 2 A •P O t* E4 .O © s s p u « © 8 © o ,0 *? P 0O © +» 0 © © to) p P o *0 O >0 OvB © O 0 « r t •H p O rt O 0 «H O E4 O JO . 0 "I3 O *H 0 BH 4» •© f © ©& >5.0 ,© » 0 O *H in L© ,H O .© ,© H © ,© a Treatments o o § ft O Q 0 *-1 © JD m h-oo o ini incu tn m m n • ♦ • • |CM H • • Pi © ftl 1 g •rt 10 13 & ^ to)to) m s * 0 Employed on the Dogs Used ■a u © © _ -H 0 1 ’g 0 „ •H © 0 0 * •H 29 ■P © O 0 © Pi 0 0 O -rl O O «pl «H K3 -H Pi © +» P« o 49 0 «H ^O fii aPi !rj W 'cSCO -P 0 0 « tt B wB © © © © Pi 0 © ft f a a VjQ 00 O CM a Pi Pi ft ft ft * FJ o o in in 00 < D oSj CV) ft © o O O O O *H Pi Pa • to jto O •d 4» o te a W O O CM h in CM 9 1 *H P in h • 6 • I *0 0 o © CO P P Q ©v£> D © © W >a © © Pi 0 «0 0 Q 8 & in 8 H SP sp SP SP m irfr) eo a CVJ K>0- < D O *rt 0 GO -P to CO B© PiS 0© M vS'B o o o o O O >H Pi o in Oo • * ml m o o iH ft ft in CM a CM m (M CM S to) o Q 49 © « 0 0 (h 49 0 *-• 67 B. Results Distribution of Ctffi ia Blood; 60 The distribution of Co in blood components is summarized in Table 11. The relation of the distribution to the time after the Co^° injected into the blood is shown in Figures 10 and 11. It will be noted that among the thirty-four blood samples tested, the cells contained only an average of 3,1 4 1.6$ of the total Co the whole blood. present in There was no statistical difference in the amount of Co in the blood cells in relation to the time after the Co blood throughout the period of twelve hours. 60 injected into the However, the amount of Go present in the cadmium hydroxide protein-free plasma filtrate decrease! toward the end of the experimental period. injection, the Co 60 Within two hours after the Co present in the protein-free plasma filtrate represented about I5.7 4 2,H$ of the C o ^ of the whole blood. The amount decreased to 60 8.9 4 1.8$ during the period of 6 to 7*5 hours after Co injection. Only If. 3 f 2.3$ was found in the protein-free filtrate near the end of the experi"* 60 ment. The differences between the amounts of Co present in the proteinfree plasma filtrates are statistically significant. amounts of Co By subtracting the present in blood cells and protein-free plasma filtrate from 100$, the amount of Co^® present in the protein precipitate were found to be 78.1 4 7 .7$, 91.1 4 3.14$ and 97.2 4 3 .5$ at various times after initial injection. By dialysis, the diffusible Co^° present in the various plasma fractions was found to be as follows? 68 A. In the blood sample obtained at one hour after Co^° injection. In Plasma In Plasma Piltrate After dialyzing of 8 hours 21$ 714$ After dialyzing of 30 hours 31$ In Protein Precipitate 9$ B. In the blood sample obtained at ten hours after Co^® injection. In Plasma After dialyzing of 22 hours 2.3$ In Plasma Piltrate 100$ In Protein Precipitate 23$ Bate of C o ^ Absorption from the Peritoneal Cavity and Its Hate of Elimina­ tion from Blood When Injected Intraperitoneally: 60 The rate of absorption and the rate of elimination of Co , in terms of half-time, when injected intraperitoneally are presented in Table 12. These data were obtained graphically from a semilog plot of the blood levels for the first four hours after injection according to the method of Berlin and Slri (1951). 60 The half-times for the removal of Co from the blood in two dogs were 13 and 12.5 minutes and for its transfer from the peritoneal cavity to the 60 blood in the same dogs were 135 &nd 195 minutes when Co was given as cobaltous sulfate. Both rates were much faster when an equivalent amount of Co^® was administered in the form of cysteine-Co^ complex. In the latter case, the half-times for removal from the blood and from the peritoneal cavity in two other dogs were 12 and S minutes, and 52 and minutes, fjr\ respectively. After four hours, the blood Cow concentration leveled off and remained relatively constant up to at least 20 hours. 69 Reabsorption of Co 6o from the Urine and Bile Samples: The data are presented in Table 13. When inorganic C o ^ was injected into the lumen of the gizzard of three-day-old chicks, the intestinal recovery, one-half hour after injection, was 70.8 ♦ b,yjL of the injected dose. 60 Corresponding figures for the recoveries of Co injected as urine were 38.6 * 2 .3 , 37.I + H.6 and 1+0 .9 ± *+.3# with an average of 38.9 + U.l$. Similar recoveries from bile samples were 1+1.1 + 3.6 , U5.I ♦ 3 .5 and I+5 .5 * 2 .9$, respectively, with an average of i+3.9 + 3 *9$* 3&e difference in recoveries between inorganic C o ^ and C o ^ from urine or bile samples is highly significant. However, there was no significant difference between the recoveries from urine and bile samples or from the samples obtained during different periods. It will be noted, accordingly, that the half-time of disappearance of Co^® from the intestine was longer in the group which fy\ received inorganic Co than in those which received urine or bile samples. Statistically, there was no difference between the half-times of the df\ intestinal disappearance of Co from urine and bile samples or from samples collected during different periods. Paper partition chromatography using radioautograms to locate the spots containing Co fin 60 was employed in an attempt to identify the forms of Co in urine and bile. There were no conclusive findings. Although the inorganic fin Co accounted for a large majority of the radioactivity in both the samples, additional radioactive component or components were present in both the urine and bile samples. In these experiments, Co^°-labeled vitamin B]^* standards moved approximately one and one-half inches whereas the standards of inorganic ♦Kindly supplied by Dr. C. Rosenblum of Merck and Co., Inc., Rahway, New Jersey. 70 6o Co remained at the origin. The other radioactive component or components moved distances varying "between one and one and one-half inches from the origin. However, in no specific case was it possible to conclude that more than a very minute trace of the Co^° in urine or bile samples was in the form of vitamin Renal Clearance, Tubular Reabsorption, Hepatic Clearance Rates of Urinary Excretion and Biliary Excretion of C o ^ : 60 Renal clearances of plasma Co in sodium pentobarbital anesthetized dogs are summarized in Tables 1^ and 15. As shown in Table 1^, between two and two and one-half hours after a 60 single injection of 20 micrograms of Co per kg. body weight, the renal clearance was found to average 26.9 ♦ 6.5 ml. per minute per square meter of surface area. In the 20 urine collection periods, the average urinary excretion of Co^® was 0.99 ♦ O.25 micrograms per minute. tration in plasma during these periods averaged The Co^® concen­ .50 * 5 ^ 6 micrograms per liter with a glomerular filtration load of l.kk -t O.Hl micrograms per minute. Prom the difference between the Co^® load and the rate of urinary excretion, the tubular reabsorption was calculated to be 69.5 ♦ 5 *5$ load. These five dogs had an average glomerular filtration rate of SS.M- 4 16.2 ml. per minute per square meter of surface area and an average effective renal plasma flow of 225*6 ± 9U.7 ml. per minute per square meter of surface area. The filtration fraction was O.U3 + 0.1^. The average tubular secretory mass for PAH (TJ was 1.86 4 0.9^ mg. per minute. Additional data for renal clearance, obtained at 6-ji and 10^-12 hours after the initial C o ^ injection are summarized in Table 15. It will be noted 71 that the renal clearance of Co 12 hours* duration. 6o decreased during the acute experiment of Between ■J-2 hours after the Co^® priming injection, the average renal clearance was 21,0 ml. per minute per square meter of surface area. It decreased to l1!.8 ml, per minute at 6-7^ hours and further to 11.5 ol. per minute lOg-12 hours after the C o ^ priming injection. Accord­ ingly, the calculated tubular reabsorption increased from an average value of 71.7$ at the beginning to 8^,1$ at the intermediate time and further to 88.5$ toward the end of the experiment. Both the glomerular filtration rate (Ccr) and the effective renal plasma flow (Cp^jj) increased with the result that there was little change in the filtration fraction during the entire period. The tubular secretory mass for PAH (fm) showed a continuous increase in one dog and fluctuated in the other. and filtration load increased slightly. fin The average plasma Cow concentration C/\ The amount of Coow recovered from the urine decreased with time. Renal and hepatic clearances of blood Go^® (based upon the whole blood C o ^ concentration) in six other dogs are summarized in Tables 16 and 17. It will be noted that the renal clearance increased rapidly and reached a maximum of 69 to 133 ml. per minute per square meter of surface area (39 to 67 ml. per minute per dog) between one-half and three hours after a single injection and fell gradually to k to Jl ml. per minute per square meter of surface area (2 to 17 ml. per minute per dog). fin In case of Co1^ infused con­ tinuously, a maximum renal clearance of 6l and 105 ml. per minute per square meter of surface area was reached within two hours after the priming injection and fell more slowly toward the termination (Table 16). Hepatic clearances showed an intial rapid increase and then leveled off to about 3 to 7.5 ml. per minute per square meter of surface area in both the group of dogs which received 72 a single injection and in the group which received a priming injection followed hy continuous infusion of Co^° (Table 17). The renal and hepatic clearances of whole blood C o ^ of the dogs which received a single injection are shown in Figures 12 and 13. Dog Ho. 3 was tion. a satisfactory prepara­ A peak renal clearance of 53 ml. P®* minute was reached minutes after Injection but the flow of urine and bile nearly ceased about three hours after injection. The hepatic clearance of this dog remained below 0 .5 ml. per minute. The ratios for clearances of Co 60 in dogs receiving a single injection to that of dogs which received a constant infusion are also presented in Tables 16 and 17. The ratio of renal clearances was found to be 0.86 at one hour after C o ^ injection (Table 16). This ratio declined consistently to about 0 .3^ at eight hours after injection, with a half-time of about four and one-half hours. On the other hand, the ratio for hepatic clearances was 1.31 at one hour after injection (Table 17). This ratio reached a maximum value of 2.06 at three hours after injection and declined to O.98 at eight hours after injection. The half-time of this decline was also four and one- half hours. 60 It will be seen in Figures 1*+ and 15 that the blood level of Co declined rapidly in the first ten minutes after a single intravenous injection of inorganic Co^. Thereafter, a fairly steady fall in blood levels took place. Eadioactive Co^® was detectable in both urine and bile samples within ten minutes after injection (Tables 16 and 17). Figures 16 and 17 showed the Co^° concentrations in urine and bile, respectively, during the entire experi­ mental period after the single intravenous injection. Maximal urinary 73 concentrations of U.5 to 30.3 micrograms per ml. were usually reached within the first hour. In the most rapid case (Dog No. H), the peak of 30.3 micro­ grams per ml. was attained in 20 minutes. Except in Dog No. 3 , urinary Co^° than dropped to levels between 0.7 and 1 .7 micrograms per ml. at the end of the experiment. Concentrations in bile increased rapidly during the first hour and then more slowly until the experiments were terminated. Pinal levels were between O.U and 1 .3 micrograms per ml. of bile, about the same as the final concentrations in urine. The rates of urinary and biliary excretion of Co^® in dogs receiving a 60 single intravenous injection, in terms of micrograms of Co per minute, are summarized in table IS. The rate of urinary Co^° excretion declined in much the same way as the urinary Co^® concentration. However, the maximal rate of excretion was not reached until twenty minutes to three hours after injection. It then declined toward the end of the experiment. biliary Co The rate of excretion continued to increase during the first half of the experiment and dropped slightly during the last half. Prom Table 1 9 , it will be noted that between *10 and 70$ of the injected dose of Co^® was recovered in urine plus bile during the course of the experi­ ments in which a single intravenous injection was given. A large majority (nearly 90$) of this was recovered during the first six hours. Only one- seventh to one-tenth of the total recovery was found in the bile. In Dog No. 3, since the flow of urine and bile nearly ceased three hours after injection, a total of only 23$ of the injected dose was recovered at the end of six hours. Blood concentrations, rates of infusion, rates of urinary, biliary and total excretion of Co in dogs which received a priming injection plus a constant infusion of inorganic Co^° are shown in figures IS, 19, 20, and 21. 7* 6o Blood concentrations of Co In three dogs (No. 6 , 7 and 25) which received an infusion at constant rates (between varying dogs from 0.627 to O.39O microgram per minute) increased very slightly during the entire experimental period of twelve hours. In Dog No. 2b, there was a very slight decrease in blood concentrations of Co^® during the same time. 60 The rate of biliary excretion of Co increased in all cases except in Dog No. 6 which showed a slight drop during the last two hours of the experi­ ment. The rise in the rate of biliary excretion continued throughout the first half of the experiment and reached a plateau four to seven hours after the priming injection of inorganic Co0^. There was a fairly constant rate of 60 urinary excretion of Co beginning half an hour after the priming injection in three of the four dogs (Dogs No. 6 , 7 25). Two of these three dogs showed a slight drop toward the end of the experiment. In the remaining dog, Dog No. 2b, the rate of urinary excretion fluctuated and declined slightly during the course of the experiment. In this dog, there was a similar slight decrease in blood concentration of Co^®. 60 The rate of total excretion of Co paralleled the rate of infusion in Dogs No. 6 and 7 . However, C o ^ was retained in Dogs No. 2b and 25, since the rate of excretion was somewhat less than the infusion rate. Tissue Distribution of Co 60 in Dogs? Twelve hours after inorganic Co^° was injected intravenously, a high concentration of Co^° was found in the liver, intestinal tract, kidney, urine and bile (Table 20). The Co^° activity in each gm. of liver (wet weight) was found to be about 2b times as great as that found in each ml. of plasma. The corresponding ratio for the intestinal wall or for the intestinal contents was 2 to 7 , for the bladder urine, kidney or bladder bile was 2 to 3«5* Except 75 in the first loop of small intestine, the concentrations of Co^® were higher in the contents than that in the wall. The concentration was about the same in the wall and in the contents of the caecum and lower in the contents than in the wall of the large intestine. When Co was injected into the lumenof the second loop oftied small 60 intestine, only about 50$ ©f the injected Co was absorbed within twelve hours. As in the case of intravenous injection, C o ^ was concentrated in the liver, in the intestinal tract, in the urine, in the kidney and in the bile. However, the concentration ratios of tissues to plasma were only 10.5 times in the liver, 1 .5 to H times in the wall or in the contents of small intestine and of caecum, 2 times in the urine and in the kidney, and about one time in the bile and in the large intestinal wall or contents. Plasma concentration in the dog which received anintravenousinjection was about twice as high as in the dog which received intraintestinal injec­ tions. In both cases, the spleen contained a smaller amount of C o ^ than in plasma, when computed on an equivalent wet weight basis. The total C o ^ recoveries from extracellular fluid at the end of twelve hours after injection were the same, e.g. 5 .3$ and 5.1$ for the dog receiving intravenous and intraintestinal injections, respectively. These calculations were based 60 upon the plasma concentration of Co and the assumption that the extra­ cellular fluid is 25$ of the body weight in the dog (Greenberg et al^, l ^ a ; Winkler et al., 19^3)• 76 TABLE 11 60 Distribution of Co in Blood at Various Times After Initial Intravenous Injection Time After Co®° Injec­ tion, hr s. Ho. of Samples 0 .5 - 2.0 19 6 .0 - 7.5 7 10 .5 - 12.0 Mean In Blood Cells 60 Co Percent of Whole Blood In Plasma In Protein-free Plasma Filtrate Protein Precipitate 6 .2 ± 7.3* 15.7 ± 2.k 78.1 ♦ 7.7 0 .0 ± 2.9 8.9 ± 1 .8 91.1 + 3.^ 8 -1 .6 ± 2.7 k.k * 2.3 97.2 1 3*5 3^ 3.1 ± 1.6 * Mean + standard error. Radioactive measurements were made separately on whole "blood, plasma and protein-free plasma filtrate as indicated in the text. Conversion of recorded counts to the actual amount of Co present in each sample was made hy comparing the sample with its standards. The following formulae were used to compute the partition of Co in the blood. 1. a s t-p y - H?r> x 100 where A B C W P y Her 3 3 * « s s = L S i*s L 2> B = * 100 3. C 2 100 - A - B Percentage of present in the red blood cells Percentage of Co®0 present in the protein-free plasma filtrate Percentage of Co®° present in the plasma protein Amount of Co®0 in whole blood (ug/ml) Amount of Co®° in plasma (ug/ml) Amount of Co®0 in undiluted protein-free plasma filtrate (ug/ml) Hematocrit 77 2ABL2 12 Half-times for the Absorption into the Blood and Removal from the Circulation of Co°0 When Administered Intraperitoneally as Cobaltous Sulfate and as Cysteine-Cobalt Complex Dog No, Blood T 3, Min. 2 Peritoneal Min. 8 13 135 9 12.5 195 Cobaltous Sulfate 10 12 52 11 8 1*5 Cysteine-Cobalt Complex Ii 78 TABLE 13 Co u Recovery (Per Cent of Injected Dose) from the Intestinal Tract and Absorption Half-times Hfhen Injected as Urine or Bile T A 13 Disappearance from the Intestine, Min. Amount of Co60 Injected ug. Intestinal C00O Recovery t Co^°S0lj. 0*813 70.8 ± 4.3* 62.5 4 13.3 0 - Hour Urine O.606 38*6 i 2.3 21.9 i l.U U - 8 Hour Urine 0*596 37.1 $ **.6 21.2 ± 2.5 g - 12 Hour Urine O.U46 U0.9 & K 3 23.5 4 2.6 38.9 i U.i 22.2 * K Z Injection Mean 0 - k Hour Bile 0.350 1*1 ,1 1 3.6 23.5 t 2.2 k - 8 Hour Bile 0.532 **5*1 * 3*5 26.3 i 2.U 8 - 12 Hour Bile 0.618 **5.5 * 2.9 26.6 4 2.2 ^3.9 t 3.9 25.5 4 U.g Mean * Mean t standard error tf J§ m l/m in • < oxn © <©• • .=* H CO ri si to • -©• m in • o m • « VO VO CO aft !-■ • K> f— VO « m i— o • m VO VO ri • I'­ Ve in m • • . • o vo VO • o cr> m • ri ri O • ri m cr> • o ov in ov co « « 3 V © © *r* +» tP. Pt 1 © Ijf © u tt © *2 28 :« r i Vi O Vi © • . 9 TJ o o •H CO h © VI Pi O © Ft © O •rl *rl O © iH ri © O § Jl © Fi «© © *rl a s w § ® V o l. © » o o o VO 1 © •ri a § *» © Ft *H p=i © % fi t s 9 g • F i ► ■£ «H Ft © •ri a a Pi if a -p Ft O © to Fi O m Vi © u 1 m CO • o CO to • o VO • O o ♦ • ° CO m • ri ,©■ CO • o CTt in • o to a> • m Jt N CO • iH cn to • o ri CO • CO in • ri cr> m • ri in «©- VO o> • CO m t— CO • m m $• £ • o VO m • o J? • o • H CO m • CO € ri • O CO • to K> CO K> » o in in • o r*H 4 K> m in • o in CO * ri • to cn • ri # © •rl a 3 ? P«U O a CO • H in rt jt 0 CO to CO § s Pi Vi o a • O i Load © © fi ► A t C one, IS tD*H *» 79 ri vo r— m CO • CO ^t- CO • « to VO to r i •M COf t i § Fi © a pi VI Vi 0 © 1 O © o a § © ri cn s o • Fi • o in Vi •© © © •ri • F* o © s© Pi to • © o R !=5 © to • VO in 5 m m • •s H K> o> a -=t CPy m o • CTi to © •a I Fi O O CO ri K"> ri VO ri to ri CO © © as P4 § Pi O 80 |M s • 0 • oca o 1 ~© a d vo 18 5 ’2 "(A rH *o h• rH rH 8 rH • in (*“ H £ VO • CO CO CO • CO VO r— in • rH l^VO • rl VO in • rH o> in • o H h• r4 rH IT i • o 8 o • O VO CM • O r4 3 K> in • o rH o> • o o in • o m rH • O VD CO o • CM rH rH • CM VO • rH in CO « o CM cr> • rl rH VD • rro rH O ■ CO m O VO • I-— CM CM in * CM CM CO m • o VO }? • O 1— O ♦ rl iH o « rH CM CO • ft rH in • H rH rH • 8 1— • rH r- in • CO CO in CO • rH co o • rH £ • rH • rH * o CTi m • o CM m • o CM CM • o CO in • o m m • o m VD • o • o S' • CM ft rH rH o • CM CM cn ♦ rH VO O • O m • in m CM VO • rH m 9 h o d © © <2 *p* (0 C\J o • CM CM rH S pj H *> • "8 • *rl *H ©CO © ft .rl «r? t © d *d p © 5 a £ I • CM # o S? o 4» « d 0 « « S 5 ° h • s -p d UCN © ft p d • li a • 3 ao m Vi o d •d o o a "H to rH o• ft • CO N- in in • rH o o • CM s • H in ft • CM oo CO • rH s i f ^co- d «rt ,a 5 & % © d d fi a SI ft VO m CM VO m CM • r— VO rH CM • CO VO CM CO $ m to • Is " oo rH iH « CM in CM rH • CO 4» © © d O Vi o © p CM © © CM 53 • •^ --0 d * OW o d •H in a 5 & I"— • cn CO ft • CM O rH in • h- O • CM rH S • VO if • o rH in d f t• ar> cr> r— • 0s! O'! o • r- h- vo • f t a> rH • rH O rH in • r~- O • CM rH s if « ft d © +> *w a 0 • 5 ^ ■ s s a d o a! © ft 1 o d d d 4* © *H d id d *rt O P -ri © P •ri © 4* © aJ ► * o • ■a 3 3o 4» •d 4-i a o « l a *—« Cc0 s 3 d § rH •ri O C0 • • CM 1 in • o »d" CM o • CM if • o in CM in » i*— S ♦ VO o • CM rH O • CM • O rH • O & 3 CO © 3 • VO s t « O « © © clearance of PAH * P o VO o o 1 O £ Renal 8 £ •“ •H © » 0 d O O CpAH a co hi— -co o crvinm CTvrH £ - Q V 0 3 in in rH rH VJ03 VOV D 3 Is3 vo 3 in m m 3 i t 3 3 m3- mmcvj H d^ • rH avK O J- rH OVH CU rH I—CVJ KVCVJVOOin rl • « • « • • » • • • • • • • • • • s w 3- o co in t-m S— C V J vo in in co ir \o 3 o Is-co r— 3 invo covo o covo vo n-3 in m 3 mcu 33 rl 0 **^. o p cd »h* .st t-co r*-co co a\ cvj tr\3 -ovo r^-Is -»h o • tninvo3 - vo vo in in in ir jet\=t3 mtn 0H■<• { « 3 incTVrHVOCOCOCOtOrH UT\ 3 3 0 K» • • • • • » • • • • • • « JTvCO l«r\«H K\VO C OVO C V Jo — CV) KM in (-4 K \3 Cr3 " m CVJ CVJrH rH rH rja H H w o d p -3 vo NCVJVOOITvCVJKVCVJCVJ Is- CTVCO ••O• • • • • • • • • ♦ • • ■c rw o o Is—cvj r— m 3 co v o i n CVJ i n v o W d - CVJ rH rH rH d *4 VO C V JCOvo •rt • in r -in • • KV (to d •rl 0 C O • • ♦ • • m3 KV»H C V J ocn^r vo3 m c -in f—rH rH O • • • • rH C OinrH 3 C V JH • • * O K M "- m 3 VD vo C V J m rH OV O 0 d4 r l• t*-covo ovomh 103 co t + • « » • • • • • « co r^-aM*—co o \m m f—q 3 rH rHVO KvmCVJfH m a ca M k c v j ■a- (to d •H o p C TtC TvC V JC V JKVKVHd H in a S 3 tiff o •35 (to a •H o p o 0 o fc © ©O a 4»Q5 Vj O M o • • • • • • • • • • NHOOVCVIOVI^OS C V JH H KVC V JrH rH rH rH • » •3• a•\ 3• 3• •m c•o mvo m “1 C V JVO (TVrH KMnOf'-CO H VOC V Jm CVJ rH rH VO IC VKVC V JVOVO O KVVO • • « • • • • • « o o r—tr\ r—o rH cr»vo f— |H in CVJ CVJ CU rH A © d **•? O H M clearance of whole blood Co © © O «8 O rH CVJ ^ SJ?rH C VJr » \i invo Is—COCFVtH Cco • Renal Vi I O » hA u> • * • • • • • o o o o o o o o 82 § -r 3i ® ■ h rH ri •P o M o O rt ri rt o a •rt -P CO o rH mVO K VM in O 60 KVt O »,=»• H ri O V • • « • • • • • H r t Wr t r t H H O ® •H o •rt o* T? ri o -P O l-l o »8 o o & m ft ft dO ® ® o 08 3 > °v o Si i n w O -ri- m *• m•m•v o•v o•v o• do• c•v w• irvw • •j • » • • • « O O O rH rH rH rH CVJ rt -ri ■rl a I ® &"» .0 W> » g o nijt1^—-CVJ A SP°w I— CtD f i ­ ve dfl o p • • • • • • • • CVJ CVJ K M f\N *N > V O i n e < ri 4 rt • h -»cwu h a ------- St rH K V N K V H IT V ri Jt h a ja VO in in in v o cvj o m o j t H cvj o O -ri- m h - m c \j^ t o cvj o o O r H C V ic v ic v jc v i m m i— i n i n in v o a - in ft -< rt • V J rH KVOVCVKWO in CO C V JrH S w O S ' rHVO O rH C h-*cu o o o o h h h h c v j k v invo invo mri- m h a f l 'C , ri -4 m m 0 xa A) a p © *rl I § E-* H *d H <8 f l O Q ■P 8 VO a ® o ® V O 00 o 0 rH rH ri O O •rt o o c v iin m o m in W M W H • U 9 rt . S.S AS? ri -4 -d M • r®i f8t do ri ^»-h ® rl H a 8 o O ri o VO o fi |ri fe 80 o ftvo o ■P © . . ri o FQ -H «H 0 *0 o jf a g l ! CU dO 3 ^ dO » o • ri O M O PQ .s i cvj cvj m • « • • o o o o o o • • • • o o o in m o o m CVJ o o o o ri £ if m CVJ H rH O C T V O C V J i n O r H o rH m tVJ CVJ m v o OVCO f^ -vo CVJ h -c o cvj r— m i n r n c r v o i n o o h h h c v j o o o • • • • « • * • • • m m d r^ t m cvj m c u in ir v r i- m r i* o • • • • • • • • • o o cvj m j t j a a a 8 o p i o cvj • • o 1— CVJ ft KOjt mri- 8 rH a ri «< ft P(P rH m • • o o ri •rt rt ® « ° o ■s-a CVJ o fl •d ovvo m n w « • • • • • * • • • • • • • • • m m r i r i cvj m m m m oooohcvjcvjcvj ts s,gy o « • Biliary da o ft h- (5 tl m o i~ -© v o o m in v o rH i*-c u v o rH in • • • • • • • • » • • • • * • • o o h cvj^ j- ^ - in v o r~- ovco m r» -v o v o n - 8 8 o -ri- -=t* m p cvj m o v o ovft-h-co jj- o o «h m m m m m m ft ® ®o £ a 0 -PVD S ft o 3 5 e-i *4 o H -P o o in cvj & m ^t h cvj clearance of whole blood Oo^G 33*° m ^ t- m v o c -s o o m h CU Injection «3 a o -=tw •H © M W {4 8 o • o i n v o CU c rv rn m m UC UC UrH-----CU CU m C CU CU CU o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o m H HU) OV© o o o o• 8• 8• 8• o o o o *© 888 • • • in v o m in o m c u v © c o p w > t n t ' - u o *h < U -= f CU C U j J . * - t f \ d - m m o• o• o« o• o• o• o• o• o• o• o o o o o o o o o o ao •rt m • • • • • • • • • • • o o o o o o o o o o o j" _ w tnmto r— -----------O O O_ H W I ^ I O m — m Cu rl BO i n CU CU .=}- CU 00 BO m j - V O H m C U CVI m m O O O O O O O O O O O s £ o £ ^jr j± cu m m c u . s t * ®o i n 0 K V 4 1 i n i n u r v r f -=£ CU of Co&> After The Bates of Urinary and Biliary Excretion 18 Single Intravenous fa TABLE H O O I© o o• o £ m in • o 8 o o o o o o o o • • • • • • • • • o o o o o o o o o ovo into o cu © cr»vo O^UVa-TKsf ftl W rl rt o o • • • • • • • • • • • o o o o o o o o o o o c u v o co K V 4 - t— j* h i n n w u ) ^ h j h o w vo3 - U K V W H H O O O O O ft m o o ©o © B z? g s, 8 fa 44 in o O o in rH cu m=i- invo i—bo o vrH o «h cu m rH rH rH rH cu m=t- 9EAB1E 19 8k Urine and Bile Volumes and Co^O Recoveries (in Per Cent of Injected Dose ) After a Single Intravenous Injection DOg No. 1 21.4 Body Weight, kg. Duration of Exp., hrs. U*5 3 13*5 k 9*5 9 8 7 5.1 3*6 5.8 3*3 3*1 6.8 15.0 29.0 Volume, ml. 7*3 5.6 1.9 k.8 Co60, % 0.1 0.6 0.1 0.6 Volume, ml. 77.8 56.5 9*1 21.2 C06O, i» 32*9 **M 22.6 55.2 Volume, ml. 3*3.9 30.3 7.0 19.3 2.1* 6.1 0.6 5.8 89.8 73*9 9.^ 32.5 36.9 60.9 23.2 60.1 50.8 38.2 7** 27.0 k.Z 7*8 0.6 10.9 Volume, ml. Urine At End of 1 Hour 2 Co^0, $ Bile 13 Urine At End of 6 Hour 0 0 Bile Volume, ml. Urine Co&>, At End of Exp. + Volume, ml. Bile Co60, $ *5 TABLE 20 Tissue Distribution of Co^O in 5^0 Dogs Twelve Hours After Injection. The percentage recovery in extracellular fluid was computed from plasma activity as indicated in the text. Cone, of Co6°, ug/mg, Wet Wt. Dog No, 22 Dog No, 23 Injected Injected Intravenously Intraintestinally Tissues Blood Plasma 0 .0101* 0.00*42* . *.0152* 0.0079* Extracellular Fluid 5.31*** Urinary Bladder Urine 0.029S* Gall Bladder Bile 5.12** ... .. 0.0159* 0.055^ o.ooss Liver 0.3529 0.08*42 Bight Kidney 0.03S0 0.01*46 Spleen 0.0125 0.0033 Wall 0.0327 0.0175 Contents 0.0320 0.01*40 Wall 0.0 ^ 1 Contents 0.0502 Wall 0.0652 0.0110 Contents 0.1062 0.0310 Wall 0.0353 _ 0*0166 Content s 0.0337 0.0153 Wall 0.0315 0.0069 Contents 0.02*48 0.00S5 First Loop of Small Intestine Second Loop of Small Intestine Third Loop of Small Intestine Caecum Large Intestine * ** - 149.6** ug per ml Becovery of Cofc0 (per cent of the injected dose) in the Blood Cells S6 20 Percentage of Co 0 20 2 k 6 8 10 12 Hours Figure 10. The Amount of Co60 Present in the Blood Cells at Various Time After Initial Intravenous Injection. 20 10 Percentage of Co Present in the Protein-free Plasma Filtrate S7 2 b 6 8 10 12 Hours Figure 11. The Amount of Co60 Present in the Protein-free Plasma Filtrate at Various Time After Initial Intravenous Injection. 88 60- 40- X------ X O O O / »■» I H l> ^ a — ..._..£ 0 O & 3 • I - 0 .002Hours Figure 19. Rate of Infusion, Blood Concentration, Urinary, Biliary and Total Excretion of Co6 0 in Dog N o . 7. 96 1.0 - Total excretion Rate of infusion 0.5 0.30.2 - ug. per Minute Urinary excretion 0 .1 - Biliary excretion 0. 0} 0 . 02. Blood concentration 0.01 0.003 10 Hours Figure 20. Rate of Infusion, Blood Concentration, Urinary, Biliary and Total Excretion of Co60 in Dog No. 2h. 97 Rate of infusion Total excretion 0.2 _ Urinary excretion ug. per Minute 0.1 0.03 - Biliary excretion 0 .0.2 Blood concentration 0.01 0 . 003. 10 Hours Figure 21, Rate of Infusion, Blood Concentration, Urinary, Biliary and Total Excretion of Coso in Dog No. 25. 98 80 • • 69.81% • 70 • Renal Clearance, Ml./Min./4Y[3S.A. ho 60 • 30 27.01 Ml ./fain,/H*S.A. •- • 20 . 1---- — 1 2 3 ---1----------------- 1----------------- 1----------^ 6 ° Urine Volume, Ml./Min. Figure 22 Relationship between Urine Volume, Tubular Reabsorption and Renal Clearance of Coeo (Data from Dog No. 18). Tubular • Reabsorption wt. 99 C. Discussion Distribution of Ckffi in Blood and Probable Protein-Bound Forms; Within twelve hours after intravenous injection of Co^°, the blood cells contained less than 5$ of the Co^° present in the whole blood (Table 11 and Figure 10). This suggests that Co does not penetrate the red blood cells or penetrates to only a very minute degree. This result is in contrast to the findings of Braude et al^ (19^9) in pigs and Cuthbertson et aL^ (I95O) in rats following oral administration of Co^® in the feed for periods of ko to 90 days. These authors reported that up to 80$ of the activity in whole blood was present in the blood cell fraction. This high level in the red blood cells may be due either to a slow penetration over a long period, or to actual incorporation into the blood cells during hematopoiesis. From the data of the present experiments, the second mechanism would appear the most probable. 60 It will be noted that the initial blood levels of Co were in the neighborhood of 0 .1 microgram per ml. after a single intravenous injection of 20 micrograms per kg. body weight of inorganic C o ^ (Figure lU), This indicates an immediate dilution of the injected dose by about 20$ of the body weight. In other words, within the first few minutes, the tracer was no longer confined to the plasma but had mixed rapidly into the entire extra­ cellular compartments. The blood level then declined rapidly for about ten minutes, indicating either removal by specific organs or entrance into tissue cells generally. Cobalt 60 bound to plasma constituents would, of course, have been counted in the blood. Therefore, the rapid removal rate must be due to loss from the plasma of "unbound" forms of Co^°. 100 Seibert et al^ (I95O) have suggested that cations injected into the blood stream may form complex, colloid-like particles. It has also been reported that as much as 75$ of the Co^° in the whole blood of sheep is in the bound and vitamin B^-like forms seven days after oral administration of inorganic 0o^° (Monroe et al., 1952b). 60 That the Co° may be in bound form in the plasma of the dogs studied here is indicated by three observations. First, less than 20$ of the blood Co ^ can be recovered from the protein-free plasma filtrate within two hours after injection (Table 11 and Figure 11). If a majority of the C o ^ is bound to the plasma protein, it would be precipitated by treatment with cadmium hydroxide, leaving only a small amount of Co^° in the protein-free filtrate. Second, the renal clearance of Co 60 decreased with time after a single intra­ venous injection (Tables 15 and l6). At the beginning of the experiment, a 60 large fraction of the injected inorganic Co kidney. is quickly cleared through the 60 However, toward the end of the experiment, a different form of Co (presumably, some protein-bound form which is cleared less readily by the kidney) may be present in plasma to account for the decline in the clearance values. Third, when plasma was dialyzed in vitro, a much smaller amount of Co^® was diffusible from the plasma procured ten hours after a single intra­ venous injection than from the plasma procured one hour after injection. This is direct evidence for a change in the ratio of diffusible to non-diffusible Co^O with time after injection. If the kidney can clear only the diffusible Co^, the renal clearance should decline, as was actually observed. 60 It is not certain, from these data, what form or forms of Co are actually cleared by the kidney. 60 Inorganic Co is undoubtedly removed from the plasma. In addition, Co^° complexed with amino acids is probably cleared to a certain 101 extent. The fact that Co^O dialyzed erut from the cadmium hydroxide pre­ cipitate of plaema indicates that at least 10$ of what might he called "protein-bound" Co^° may be “available" for renal excretion. Thus, the Co^O brought down by cadmium hydroxide is in at least two forms, one of which, since it is dialyzable, is presumably capable of being cleared by the kidney. It is possible that the dialyzable Co^O was merely adsorbed on the protein precipitate and subsequently eluted in dialysis by exposure to the large volume of external solvent. In the protein precipitate of the blood sample obtained one hour after Co^O injection, 9$ of its Co^® was diffusible in a dialyzing period of eight hours. Since the C©^® in the protein precipitate represeats 78$ of the whole blood C o ^ in the first two hours (Table 11), it may readily be com­ puted that 72$ of the whole blood Co^O ig non-diffusible (78$ - 9$ * 78$) • Similarly, in the blood sample obtained ten hours after injection, 23$ of the protein precipitate Co^O was diffusible. About 97$ of the whole blood Co^® in the sample collected ten and one-half hours after injection was precipitated in the protein fraction (Table 11). Thus, about 75$ of the whole blood Co^O was non-diffusible (97$ - 97$ x 23$), a figure identical with that found at two hours. This means that the total amount in the non-diffusible Co^° form in the blood apparently does not change with time after a single intra­ venous injection. Boulanger et al. (1952) have reported that cadmium hydroxide brings down all proteins and polypeptides in the urine and that all amino acids remain in the filtrate and in the washing solution of the precipitate, 102 It is interesting to note here that the form of Co^° which will diffuse through the dialyaing membrane does not diffuse through the membrane of the red blood cell. As will be pointed out^Later, it appears that some fraction 60 of the Co which will not dialyze through the cellophane membrane will, however, diffuse across the glomeruli of the kidney. The turnover rate of Co within the biological system is faster when it is in the form of amino acid complexes than when it is present in inorganic form (Table 12 which confirms the general conclusion of Berlin and Siri, 1951). 60 The fact that the Co in urine and bile samples has a more rapid rate of absorption from the chick intestine than has inorganic Co 60 (Table le) suggests fsf) that the Cow in the urine and bile, or a portion of it, may exist in amino acid complexes. It has been reported that 7 1Q$ °f the total Co 60 in the urine of sheep is recoverable as a vitamin Bj^-Sdke substance (calculated over a 60 period of seven days after administration of inorganic Co )(Monroe ejb al., 1952b). 60 Both bound and vitamin Bj^-like forms other than inorganic Co were found in all tissues tested by these investigators. On the basis of these findings, it might be presumed that certain fractions of the total Co in the bile could be in some organic form. 60 Accordingly, preliminary paper partition chromatography and radioautography was undertaken on selected urine and bile samples obtained from the dogs employed in the clearance studies. 60 The results indicate that inorganic Co present in these samples. labeled vitamin is not the only radioactive form Comparison with a standard preparation of Co^°- failed to reveal significant amounts of vitamin B^g in either the bile or urine samples. The present data do not confirm the findings of Monroe et alj. (1952b) *n sheep. The failure to find vitamin 103 1^2 in either the urine or the hile from dogs may he a true species difference from the sheep. On. the other hand, the quantity of the urine or the hile used in these studies may have contained less than the minimal 60 quantity of Co detectable hy radioautographic methods. Urinary and Biliary Excretion of Intravenously Injected Inorganic C o ^ t Mar&n (1952) has summarized the fairly extensive data leading to the conclusion that parenterally injected cohalt is excreted mainly in the urine, hut in part hy the hile. The total Co 60 recoveries in urine and hile ob­ tained in the dog under the present acute experimental conditions and after single intravenous injection (Table 19) are in general agreement with those reported previously in other species (Comar and Davis, 19^7a; Comar et al., 19^6a, 19*+6b; Copp and Greenberg, 19^1? Greenberg e£ al., 19^3^» Sheline et al., 19^6; Monroe et al., 1952b), The rate of excretion (in micrograms per minute) hy the kidney was very rapid during the first few hours after intravenous injection. The total Co was much higher than that from the hile. 60 recovery from the urine 60 Since the Co in hile is reabsorb- 60 able in the intestinal tract, a large fraction of the Co recovered from hile must normally be recirculated through the body. Thus, the net rate of Co^® loss in bile fistula dogs should be somewhat greater than that in intact 60 dogs whose biliary Co passes into the intestine from which it will be reabsorbed. 60 In the hile fistula dogs, fecal loss of Co may he chiefly composed of that fraction of the intestinal Co^° fixed by the intestinal bacteria. The rapid increases both in the urinary concentration and in the rate 60 of urinary excretion of Co soon after a single intravenous injection (tfigure 16 and Table 18) indicate that the plasma Co^° rapidly filters 10k through the glomeruli. three hours. The maximal values were reached within one-half to 60 As the plasma Co concentration rapidly dropped and then more slowly declined, corresponding changes in the urinary concentration and in the rate of urinary excretion followed. However, changes in the hiliary concentration and in the rate of hiliary excretion were much more gradual. A maximal rate of hiliary Co fin excretion was not reached until five to seven hours after a single intravenous injection (Table 18 and Figure 17). At this time, the blood concentration and the rate of urinary excretion of C o ^ were far below their maximal values. Thereafter, the rate of hiliary excre­ tion of Co^O decreased only slightly. This suggests that the delay of Co^® in the liver may he much longer than in the kidney. The first appearance of C o ^ both in the urine and in the hile was five to ten minutes after intra­ venous injection. appearance of Co 60 There is, then, no greater delay in the initial time of in the hile than in the urine. However, the peak of the hepatic excretion curve is delayed about four hours in comparison with that of the urinary curve. A similar delay of phosphate (Kleiber, 1952a) and of acetate (Kleiber, 1952b) by the mammary gland of the cow has been reported. The delay in the liver is slightly less, however. The ratio of hepatic clearances of Co^G in dogs receiving a single injection to that of dogs which received a constant infusion increased during the first three hours after injection (Table 17). after. It reached a maximum value of two and fell off there­ On the other hand, the ratio of single injection to constant infusion for renal clearances in these same dogs was less than one at the beginning and declined gradually (Table 16). It should be noted that from three hours on, the half-times for the decline in these ratios were the same for the renal clearance curve as for the hepatic clearance. Finally, in dogs receiv­ ing a constant infusion, the rate of biliary excretion increased gradually and 105 reached a plateau within four to seven hours after injection (Figures 18 to 21), However, the rate of urinary excretion was relatively constant through­ out the experiment. It would appear that the kidney and the liver clear the same form of Co^°, at least after the third hour after Co injection, although these two organs handle this tracer at a different rate. This conclusion is based on two Cj\ types of findings. First, the Co from urine and bile was absorbed at the same rate by the intestine of young chicks (Table 13). Second, half-times for the decline in the ratios for renal and hepatic clearances in dogs receiving a single injection to dogs which received a constant infusion were both about four and one-half hours. In each case, one would expect different Go half-times if different forms of Cow were cleared by the kidney and the liver. The biliary excretion rate for Co^G in four dogs which received a constant infusion reached plateau values of 0 .0^5 to 0.0 6 microgram per minute within four to seven hours after injection. That these values were reached and then maintained for seven to four hours infour different animals indicates that some maximal, or saturation output rate exists. In experiments in which fluctuation in the perfusion rate occurred, corresponding variations in the rate of urinary excretion of Co were noted (Figure 20). However, the rate of biliary excretion was not influenced. Go A low rate of total excretion resulted in retention of Coou in two of 60 the four dogs which received a constant infusion of Co . The blood concen­ tration of Co^G increased in one of these two dogs and declined in the other one. Since no tissue sample from these dogs was analyzed, how the retained Co^° was distributed in the body is not known. 106 Renal Clearance and Tabular Reabsorption of C o ^ » It has been noted in a previous section that a large fraction of the whole blood Co experiment. So is in some "non—diffusible" form or forms throughout the Since the total blood C o ^ decreased from an initial concen­ tration of 0 .1 microgram per ml, to about 0.01 microgram per ml., a reduction 60 of ten times, at least a part of the "non—diffusible*1 Co must then have been "available" to the glomeruli of the kidney for clearance. Since the "non-diffusible" form or forms of Co^° in the blood may be in some "protein-bound" form or forms, and since the protein moiety of the 6o complex is not cleared by the kidney, it appears that the protein-Co plex i8 readily dissociated. com- The rate of dissociation for this protein-Co 6ft complex is not known. Half of the plasma calcium is combined with plasma protein. Calcium and magnesium proteinates are much less completely dissociated than are the corresponding sodium and potassium salts (Smith, 1951)* Nevertheless, forma­ tion of the calcium proteinate "does not appear to interfere with the trans­ capillary movement of calcium" (Armstrong et_ al.» 1952)* A similar situation may well exist with respect to the cobalt. 60 Since the Co is cleared, all renal and hepatic clearances were cal- 6ft culated on the basis of whole blood or whole plasma Cow concentration rather than on the basis of that portion not precipitated by cadmium hydroxide. Thus, the calculated clearance values may be smaller than the true values 6ft by whatever fraction of the whole blood Cow is not "available" for filtration. In experiments of short duration, the barbiturates have no effect on renal function (Corcoran and Page, I9H3); but for experiments of long dura­ tion (more than three hours), a reduction in effective renal plasma flow 107 and in the maximal Tm for PAH have been reported (Glauser and Selkurt, 1952). The glomerular filtration rate observed in five dogs in these present experi­ ments within three hours during sodium pentobarbital anesthesia (Table 1^) fell within the normal range for the 75 normal, trained, female dogs not under anesthesia tabulated by Houck (19^8). It is reasonably certain that these five dogs were normal in the presence of mild surgical trauma and that their renal function was not deteriorating during periods up to three hours. In these present experiments of short duration (up to three hours) an average renal clearance of 2J ml. per minute (Table ik) indicates a high degree of tubular reabsorption of Co**0 , as was calculated. It is important to note the uniformity in the renal clearance values and in the percentages of tubular reabsorption in each of the five dogs (Appendix h). Renal handling of strong electrolytes has been summarized by Smith (I95I). Sodium and chloride are completely reabsorbed by tubules tinder normal con­ ditions. Extensive potassium reabsorption occurs in the proximal tubules, but resecretion takes place in the distal tubules. of calcium remain ambiguous. unknown. Renal clearance studies Renal handling of magnesium and strontium is Iodide and thiocyanate clearances are very low and influenced by the sodium and chloride concentrations. There is, then, little information in the literature concerning the handling of electrolytes which is of aid in evaluating the cobalt data. It is evea possible that cobalt might be excreted as an anionic complex ion, rather than as a cation. As has been noted pre­ viously, the renal behavior of the Co^° precipitated by cadmium hydroxide is not clear. This type of uncertainty is the present limitation to the Interpretation of the clearance data. Until the chemistry of the cobalt complex in blood is known, the true extent of tubular reabsorption cannot be accurately determined. 108 Since a certain fraction of the blood Co**0 may be in the form of acid complexes which are diffusible on dialysis, these amino acid complexes may then be present in the glomerular filtrate. Total amino acid clearances have been reported to be very low, usually less than 10 ml. (Kirk, 1936). The essential amino acids are very effectively reabsorbed by the tubules (Beyer et al., 19^6; Doty, 19^3* Goettsch et al., 19^-; Harvey and Horwitt, 19^9» Kirsner et a L , I9U9 ; Pitts, 19^3; Russo et aJL, 19^7 ? Sheffner et al., Wright et al., 19^7) • This suggests a mechanism which could account for a portion of the low clearance values and of the relatively high percentage of tubular reabsorption of Co**° in the present studies. In these experiments of long duration (up to thirteen hours), a rela­ tively constant renal clearance of Co**° was maintained in dogs which received a constant infusion (Table 16). This directly indicates that, in the dog, the surgical operation employed in these experiments does not significantly affect the function of the kidney as far as the handling of the C o ^ is concerned. 60 Thus, the reduction in the renal clearance of Co following a single injection (Tables 15 and 16 and Figure 12) must be primarily due to the changes of the renal function (including the clearance of a different form of cobalt) rather than to surgical trauma. This is directly indicated 60 by the fact that the percentage of tubular reabsorption of Co increased with time after injection (Table 15)* It has been reported that variations in urine flow have no effect on glomerular filtration rate in rabbits under controlled experimental condi­ tions (Forster, 1952). In these acute experiments, a 16-fold variation in urine flow (0.*K> to 6.57 ml. per minute in Dog No. IS weighing 9 kg.) failed in change the relatively constant renal clearance and tubular reabsorption of Co6° (Figure 22). 109 Tissue Distribution, Intestinal Absorption and "Secretion" of Ccffi: Data from the dog are in agreement with conclusions previously obtained in the chicken. fin First, inorganic Cow is readily absorbed by the small Intestine of the dog. A segment of the first part of small intestine, iso­ lated from the rest of the tract by ligation, absorbed half of the injected 60 Co from its lumen within twelve hours. absorption rate observed in chicks. This is much slower than the Second, a relatively large amount of fin intravenously injected inorganic Co gets into the intestinal tract of the dog through the intestinal wall, in addition to the amount reaching the tract through the bile. Whether this active passage of Co^° across the intestinal wall is a process of "secretion" or merely "continuous diffusion" remains uncertain. Third, Co 60 must be in some "non-diffusible" forms in the con- tents and also in the wall of the intestinal tract of the dog, since the Co fin concentration in the contents or in the wall were greater than the plasma concentration (Table 22). Considerable biosynthesis of vitamin B12 or B^g-like 8Ul:)s^ajac8 ^ias 158611 60 reported to take place in the intestinal tract of sheep after Co ingestion, anri in the tissues after intravenous injection (Monroe et al., 1952b). According to these investigators, the vitamin fin Cow in the small intestine is from 6 to 15$, and the bound Co**0 in this organ is from 4l to 53$. This bound Co**0 may be present within the bacterial cells. The fact that the contents and the wall itself in the first tied seg- ment of the small intestine contained the same low concentration of Co indicates either less binding or greater absorption of Co 60 60 in this segment. It should be noted that the bile duct empties into this loop. Hence, the increasing concentration of Co^°, both in the contents and in the wall, as 110 one passes from the first to the third loop suggests increased retention of 60 Co in the lower segments. The more activity in lower segments may he due either to some "binding” agent (for example, bacteria or intestinal con­ tents) or to increased secretion into the intestinal lumen. Unlike the chicken, the concentration of C o ^ was about the same in the contents as in the wall of the caecum in the dog. The Co concentration ratios of caecal wall or caecal contents to blood were much less in the dog than in the chicken. When one considers tissue retention ofCo^®, the liver has been reported to be one of the tissues in which cobalt is concentrated in the largest amount (Braude et_ al., I9U9 ; Comar and Davis, 19^7®* 19^7^» Comar et_ al;, 19^6a; I9h6b; Copp and Greenberg, 19^1; Cuthbertson et al., I95O; Greenberg et al., 19^+3^I Lawrence, I9U7 ; Monroe et al., 1952a; 1952b; Ulrich and Copp, 1951)- I* &®s also been known that liver is a very rich source of vitamin 3^2 la ruminants (Lewis et al., I9I+9 ; Thompson et al., 1950). However, no evidence for the biosynthesis of vitamin B^g or B^^-like substance was shown in the liver of sheep (Monroe et al., 1952b). The following points emphasize the role of the liver in the metabolism of Co 60 in the dogs first, in the acute bile filuta dogs, H to 11$ of the fin intravenously injected inorganic CoDU was excreted in the bile during a period of seven to thirteen hours (Table 19). 60 Second, a form or forms of Co other t.v>gn its inorganic form is present in the bile. two lines of evidence. This conclusion is based on (1) The absorption rate of the bile Co^° was faster than that of its inorganic form (Table 13); (2) paper partition chromato­ graphic and radioautographic separation studies revealed the presence in the 60 bile of a radioactive component or components containing Co other than in its inorganic form. Third, twelve hours after intravenous or intraintestinal Ill 60 injection, Co was much more concentrated in the liver than in either the kidney, the contents or the wall of the intestinal tract, the bladder urine, the gall bladder hile or the spleen. from these observations, it is certain that the liver in the dog plays 60 an important part in the metabolism of Co and that its physiological role in handling this tracer is apparently different from that of the kidney. The kidney apparently serves only as an excretory organ for Co . The liver, on the other hand, may be a storage organ for various types of cobalt com­ pounds, or may even synthesize certain of these compounds. Comar and Davis (19*+7b) have reported that cobalt is concentrated 100 times in the liver of a young calf than In the whole blood seventeen hours after intravenous injection of labeled cobalt. The cobalt concentration ratio of the liver to whole blood in ruminants is much higher than that found in dogs. This indicates the greater importance of liver in ruminants in the metabolism of cobalt than that in non-ruminants. It also accounts for the fact that the liver of ruminants is a very rich source of vitamin B^g (Lewis et al., 19^9: Thompson et al., 195°)• The relatively high concentration of Co^° in the kidney may reflect the excretion of this element through that organ. Since the concentration of Co^° in the spleen was lower than the plasma level, the biosynthesis of vitamin ®12 or vitamin B^g-like substance in this organ in the dog (as reported by Monroe et al., 1952b, in the sheep) remains doubtful. 112 SUMMARY 1. go Cobalt 6 0 , as Co SO^, is both readily absorbed from the intestinal tract and "secreted" or "continuously diffused" into the intestinal tract through its wall in both the chicken and the dog. Twenty-four hours after Co^° is administered intravenously or injected into the gizzard of the chicken, 6$ of the injected dose may be recovered from the small intestine, 5# from both caeca, 1# from the large intestine and 5# from the extracellular fluid. During this period, if the Co^° is injected intravenously, at least 13$ of the injected dose is eliminated in the feces and a large fraction of the remaining 70$ is excreted in the urine emptied into the cloaca. 60 If the Co is injected into the gizzard, at least 51$ appears in the feces and a fraction of the remaining 32$ appears in the urine by the end of 2k hours. After a single intravenous injection of C o ^ into the dog, between Uo and 70$ of the injected dose may be recovered in the urine plus the bile during a period of seven to thirteen hours. A large majority of this (nearly 90$) is recovered in the first six hours. Only one-seventh to one-tenth of the total recovery is found in the bile. 60 Ihen Co is injected into a loop formed at the first part of the small intestine, half of the injected dose is absorbed by the end of twelve hours. 2. the gut. In the chicken, most of the Co found in the caecum entersfrom However, under certain experimental conditions (such as complete obstruction of both the large intestine and of the caecum itself), Go^° passes into the caecum through the wallin large amounts. In the dog, caecum plays an insignificant role in the metabolism of Co^°. the U3 3* In the chicken, Co 60 is greatly concentrated in the caecal contents, in the caecal wall, and in the large and small intestines regardless of the route of administration. In these locations, it is present in some "non— diffusible" form or forms. bile of the chicken, A very small amount of Co^° is found in the bladder la the dog, the highest concentrations of C o ^ are found in the liver, in the intestinal wall and in the contents of the first part of the jejunum, in the kidney, in the gall bladder bile and in the bladder urine. The spleen contains a very low amount of Co^. Cobalt 60 passes into the extracellular fluid soon after intravenous injection of its inorganic form in the dog. ficant amount of Co^. Blood cells contain an insigni­ About 75$ of the whole blood Co^° is precipitable with cadmium hydroxide and remains "non-diffusible” on dialysis. This "non- diffusible" Co^° in the protein precipitate appears to be "dissociable" and "available" for clearance by the kidney and the liver. 5. In the dog, the plasma C o ^ quickly filters through the glomeruli following a single intravenous injection. The maximal values for the rate of urinary excretion are reached within one-half to three hours. The urinary excretory rate then decreases as the plasma C o ^ concentration drops. On the other hand, the rate of biliary Co 60 excretion reaches a maximum about five to seven hours after injection and then drops only slightly. In dogs receiving a constant infusion, relatively constant plasma C o ^ concentrations are maintained. 60 The rate of urinary Co excretion in these dogs remains constant but at a level dependent upon the rate of infusion. However, the rate of biliary excretion increases gradually and reaches a plateau at about four to seven hours. 60 The first appearance of Co both in the urine and in the bile is within five to ten minutes after intravenous injection. There is a "delay" of 114 about four hours in the peak of the biliary excretion curve when compared with the peak of the urinary excretion curve. 6* The renal clearance of C o ^ within three hours after intravenous injection averages 27 ml. per minute per square meter of surface area in five sodium pentobarbital anesthetized dogs in the presence of mild surgical trauma. About three-fourths of the filtration load is reabsorbed by the tubules, when the calculation is made on the basis of whole plasma C o ^ concentration. In acute experiments of long duration (up to thirteen hours), 60 renal clearances of Co decline due to an Increase in the tubular reabsorption. 7. In the dog, the half-time for the removal of Cow from the blood is 20$ faster when injected as the cystein-Co^® complex than when injected as Co^SOty. The transfer of Co^® from the peritoneal cavity to the blood is 3 .3 times faster when injected as the cysteine-Co^ complex than when injected as Co^SO^. 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Trace Elements in Livestock Diseases. 531-533*. 1939. Chem. Indus. Chem. Indus. 125 Appendix I The Cobalt 60 Mass Absorption Coefficient The external absorber method of Aten (Aten, Jr. 1950) was used to con— struct a self-absorption curve for Co^°. in which Co cover. 60 About 80 mg. of ashed intestines was uniformly distributed were evenly mounted on a crucible The activity was measured under the same geometry as employed throughout the chicken and dog experiments. The activities of the same sample covered hy a series of thin aluminum absorbers were then counted. The follow­ ing formula was employed to calculate the absorption coefficient (u)i where A& is the activity observed without an absorber, A& is the activity observed through an external absorber of thickness ^ (mg. per square cm.), and u is the mass absorption coefficient (square cm. per mg.). Four absorbers ranging from I.75 to 13.70 mg. per square cm. were used. Activities with and without the external absorbers and the calculated values for the mass absorption coefficient are summarized in Table 1. Cr\ Table 1. The Mass Absorption Coefficient (u) for CoDU. Thickness of External Absorber, mg/cm 0.00 1.75 13.70 Activity cps ^S.503 ^2.966 38.816 31.278 18.335 Calculated u cm /mg 0.069 0.069 0.072 0.071 126 An average value of 0.070 was taken for u» By substituting the value of u and different values of Xq into the formula given above, the activity ratios (Aa/Ajq) were calculated and Table 2 and Figure 1 were constructed. Table 2. Activity Batios (Ag/A*) of Co60 at Different Thickness of Sample, Thickness of Sample mg/car Activity Batio, $ Calculated 1 2 93.2 3 6 SI.3 75.9 70.8 66.7 7 61.7 8 57.5 53.7 50.1 35.5 k 5 9 10 15 S7.1 20 25.1 25 30 17.2 Data from Gleason et al. (1951) 91.6 23.9 76.8 70.4 64.5 59.0 5^.1 12.6 In practice, the thickness of a sample is obtained by dividing the ash weight of the sample (mg.) by its surface area (cm^). Figure 1 is entered with this value on the abscissa. The plot given as The corresponding value of the activity ratio may then be read on the ordinate. The measured activity of the sample is then divided by the activity ratio, the result being the true activity of the sample. Schweitzer and Stein (1950) reported good agreement between three different methods, including this method of Aten (1950), when used to correct for the effect of self-absorption of beta particles. computed Gleason et al^ (1951) a u value of 0.088 om£/«g for Co60 from the equation! 127 u = 0.017 where u is the mass absorption coefficient near zero thickness, and Bmar is the maximum energy of the beta spectrum in Mev. With their counting geometry, the computed value for u was experimentally confirmed. The lower i u found in the present case is presumably to be ascribed to scattered and secondary radiation. m ffl H a cn o to to © o Eh GO CM O O CM s % ‘ (ihv/bv) otc^bh Aj.XAxq.ov Curve O Self-absorption N s o 1. (M Figure xs -p © a -p © for Co60 128 129 Appendix II Methods and Calculations for the Study of Renal Function in the Dog 1. Protein-free Plasma Pi Itrate: . 1. Centrifuge the "blood sample to obtain plasma. 2. Pipette 2 ml. of plasma into 10 ml. of water in a clean 50 ml. centrifuge tube (do this in duplicate for each blood sample). 3. Add l6 ml. of acid cadmium sulfate* from a burette and mix. Add 2 ml. of 1.1 H NaOH, stopper and shake well. 3 . Let stand for ten minutes and centrifuge. The water-clear supernatant layer is used for chemical analysis. 2. Urine Creatinine? 1. Pipette 3 ml. of the final diluted urine into a 5 ml. colorimeter tube. 2. Add 1 ml* of saturated picric acid and mix. 3. Add 1 ml. of 0.75 N NaOH and mix. Let stand exactly fifteen minutes and read in the colorimeter using filter of 5^0 mu. Do this in duplicate. Reagent blank: StandWs? Three ml. of distilled water is used. Three tubes containing known amounts of creatinine (20, Ho and 60 micrograms) in 3 ml. of distilled water solution will be made. nm HuTfftte is made from the following formula: CdSOh* SHgO *3 1 H H2 SOI). Distilled water, q.s. 63.5 ml. 1000 ml. Do 130 this in duplicate. A standard curve is then made hy plotting the amounts of creatinine versus the readings on the colorimeter. 3 . Plasma Creatinine: Pipette 3 the protein-free plasma filtrate (water-clear supernatant layer) into a colorimeter tube and proceed as for the urine creatinine described above. Sometimes, when the O .75 N NaOH is added, a faint flocculant precipitate forms which must be thrown down in the centrifuge. To do this it is probably best to develop the plasma creatinine color in a conical centrifuge, tube. Centrifuge during the 15-minute color- development period, then pour the contents into a colorimeter tube for reading. As before a reagent blank and three standard tubes will be made. k. TTrine PAH: 1. Pipette 3 ml* <*£ the final diluted urine into a large test tube containing 7 ml* of water. *2. Add 2 ml. of 1.2 N HC1 from a burette and mix. 3 . Also from burettes add the followings a. One ml. of NaflOg (100 mg.$), shake vigorously. b. After standing not less than three nor more than five minutes, add 1 ml. of ammonium sulfamate (500 ag.£) c. and mix thoroughly. After standing not less than two nor more than five minutes, add 1 ml. of N(l-naphtyl) ethylenediamine dihydrochloride (100 mg.$) and mix well. k. Let stand for ten minutes and read in the colorimeter tubes using filter of 5*K) mu. Do this in duplicate. This color is stable. There is no need for undue hurry. Reagent blank: Ten ml. of distilled water is used. Standard: One tube containing a known amount of PAH (20 micrograms) In 10 ml. of distilled water will be made. Do this in duplicate. There is a direct proportion between the amount of PAH present in the solution and the reading from the colorimeter. Plasma PAH: Pipette 5 of the protein-free plasma filtrate (water-clear supernatant layer) into a large test tube containing 5 ml. of distilled water. Proceed as for the urine PAH determination described above. As before, a reagent blank and a standard tube will be made. Formulae Used in Calculations: ^cr7 1 . Ccr « ----J?cr where CCr is the renal clearance of creatinine in ml. per minute, Ucy is the urine concentration of creatinine in mg. per ml. (of undiluted urine), Pcr is the plasma filtrate concentration of creatinine in mg. per ml. (undiluted filtrate) and V is the urine volume in ml. per minute. a 2‘ m CPAH “ UPAH i>A m 7 ----PAH where CpAH is the renal clearance of PAH in ml. per minute, UPAH is the urine concentration of PAH in mg. per ml, (undiluted urine), Pp^jj is the plasma filtrate concentration of PAH in mg. per ml. (undiluted filtrate) and V is the urine volume in ml. per minute. uCo6o V where Cq06o is the renal clearance of Co u in ml. per minute, UCo6o is the urine concentration of C o ^ in micrograms per ml, (undiluted urine), 132 PCo60 Is the plasma concentration of Co in micrograms per ml. V is the urine volume in ml. per minute. U. Tm = U p V - Ccr x Pp^g x FW where 3^ is the tubular secretory mass of PAH in mg. per minute, Up^ is the urine concentration of PAH in mg. per ml. (undiluted urine), V is the urine volume in ml. per minute, Ccr is the renal clearance of creatinine in ml, per minute, Pp^g is the plasma filtrate concentration of PAH in mg. per ml, (undiluted filtrate) and the 3PW factor is taken as O.917 as suggested hy Smith (1951). Renal Clearance of PAH 5. nr = - - - - - - - - - - - - - - - - - - - - Renal Clearance of Creatinine where FF is the filtration fraction. 6. Co^° Load (ug/min.) = Glomerular Filtration Sate or Ccr (ml/min.) x Plasma Concentration of Co^° (ug/ml) 7 . Co^0 Excreted (ug/min.) = Urine Volume (ml/min.) x Urine Concentration of Co00 (ug/ml) g. 9. Co^° Reahsorhed (ug/min.) = C o ^ Load (ug/min.) - Co^® Excreted (ug/min.) . Co^® Reahsorhed Co50 Reahsorptlon (#) - ----- tz -------Co® Load 133 \R VO H m u fr o i n n - c u in B O r H i— c u r— m in v o m • • • * * • • I C V * O h• • • • • • • • • • • • cu v o eo m m cr>B O oo m n c i i n rH m # H rH C M — cu cu m A h-®o CT»BO BO r— t— k ~ — r—vo’f — (T io o o o c n c n c n o < T iO o b i © % u +3 H J=t f— § 8 in in *» o U Pi H O d MvS O rt o « H O P* % O O o ■+> o u o +3 © <6 b o © •+3 < {o H H H H H H H p i h h rH cu jd - B O .= t-jd * > o ^ t cu i h tnmVO OVOV0VOVO rHV© i— cu m v o j - KNH *H ‘f bo » cu m H• CT*M-H • r-VD • • mcu * • « •« •=r•m H [bo »— m i n o crim I s— in invo I— cu CU H ou m | r— vojd- II 1 cu i n i — rn v o . o cu m o — >O oo vo M 00Jt Jt I T i l l II t-i BO rH BO IX) f— f— CO VO IT> u 1_ vojd- r-r-w) m^d- cu rH cu cu K hrH H rH H H H H C U C U r H C U r H r H H r H r l r H H o o O• O« O* O• O• o o o o O O O O O O O O O O O • • • • • • • * * • • « • • • • o o o o o o o o o o o O O O O O O O O O O O —0 m s * r — cu h c r » m m | — KMOrl m c n v o v o cu ^ 1 ovd- I m m pnvo CU O rH co t n m v o bo n K i H v o M er»o r— r— in m m m m m m m cu cu cu cu m m m i d - m m m m c u cu cu 0 0 !0• 0* o o o• o• o• o• o■ O O O O o• o• 0• 0• 0« 0 » 0 • • ••«* • • o o o o o o o o o o o O O O O O O O O O O O in un z? i— mvovo O o o cu h • • « • • • • « • • • 1— m c u bo m c o 1 m cnm — iomr— i r i o o O H O N H H m m m m j t in ini Jd’ o v d - ^ t v p i n r— bo oo vo m m m d - ^d-^d-«st-arjt am m o o o uvd- co CO _ m m ino op m i n rH in m intrxsf invo 5 m Q O O O 8• 8 • O• O• O• O• * • ♦ • 8• 8• 8• 8• 8• 8• 8• 8• 0* 0« 0■ O O o o o o o o o o o 0 0 0 0 0 0 0 0 0 0 0 t* O *rf <8 CTiVO 00,=f to CU • • • • * « • j=)r in in !§ ‘S f— O H H N CU SOVO KNVO SO h-H S- KVt VO SOjSj-VO H V O N r^-vovo h-h- s ; e g ' g " & s - s CU f- CU^f OMT*.KSri N - O X r H V O < ru d - m r — c ri © •H r 4 pH H H t*-© v o rH r~-^t m Si r3v a£- 3K x a:?- 3m 3m c3u ;-?JP 3 'ft3 \ cu cu cu k h 8 8 8 8 8 8 8 8 8 8 8 8 0 0 0 0 0 0 0 0 0 0 0 - 0 v o m tu m m m o s o v o c u m c u • • • • • • « • • • • • m c * -m = j- s o O H O O H O i n K t^W h\jJ h- h h hh-ff\ONO h h w wH h0)W w w(H © O O O O O O O O O O O O O O O O O O O O O O O (^CT\OnH ITvVD O g\ 0 H W < o h o u ) o w m in r \i CUCUWCVJmmmmmmKV u> w O O O O O O O O O O O ! 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