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The metabolism of carbon¹⁴- labelled urea Wright, William Douglas 1952

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L<5 5 /5 ? fir THE METABOLISM OF CARBON14 - LABELLED UREA by ' WILLIAM DOUGLAS WRIGHT A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE; OF MASTER OF ARTS i n the Department of CHEMISTRY We accept this thesis as conforming to the standard required from candidates for the degree of MASTER OF ARTS THE UNIVERSITY OF BRITISH COLUMBIA Members of the Department of April , 1952 ABSTRACT Urea, labelled with carbon*-^ w a s synthesized and administered 14 to rats by intraperitoneal injection. The excretion of carbon1- was followed by analysing the urine, feces and expired a i r quantitatively for radioactivity at various times after injection, and the distribu-tion of the isotope was studied by analysis of organs, blood and car-cass. A portion of the injected urea was rapidly metabolised, approxi-mately 30 percent of the isotope being excreted i n the expired a i r after 12 hours. The highest output of C^ O^g occurred during the second hour after injection. The majority of the remaining isotope was excreted i n the urine as urea. After three hours only a small percentage of the injected carbon^ was present i n the kidney, l i v e r , and blood largely i n the form of urea. After 48 hours, approximately five percent of the carbon^ injected as urea remained i n the carcass and appeared to be fixed i n the tissues. ACKNOWLEDGMENTS This research was carried out under a grant from the National Research Council. The author i s indebted to Dr. S.H. Zbarsky for his encouragement, criticism and advice throughout the course of this research. TABLE OF CONTENTS INTRODUCTION . . 1 EXPERIMENTAL A. Measurement of Radioactivity (i) Oxidation 6 ( i i ) Counting 8 B. Synthesis of Labelled Urea 12 C. Metabolism Experiments (i) Apparatus 15 ( i i ) Rat 1 17 ( i i i ) Rat 2 20 (iv) Rat 3 v 25 DISCUSSION . . . 29 SUMMARY 33 BIBLIOGRAPHY 35 INTRODUCTION The compound urea i s the principal end-product of nitrogen metabolism i n mammals. The synthesis of urea has been shown to take place primarily i n the l i v e r by experiments i n which there was found to be complete cessation of urea formation after hepatectomy. In vitro studies using tissue slices under nearly physiological conditions have also shown that only l i v e r tissue i s capable of synthesizing urea. The actual mechanism of urea formation i s not definitely known but the theory proposed by Krebs and Henseleit (1) i s generally accepted. Ammonia derived „f rom amino acids by oxidative deamination and carbon dioxide from metabolism of fat, carbohydrate or non-nitrogenous protein residues combine with ornithine to yie l d c i t r u l l i n e . Citrulline i s then converted into arginine by addition of another molecule of ammonia and arginine through the influence of the enzyme arginase breaks down to yi e l d urea and ornithine. The urea thus formed i s excreted i n the urine. It was generally assumed that urea was excreted unchanged after i t had been formed. It has been suggested, however, that because of i t s structure, urea might be an intermediate or precursor i n the formation of creatine, (2) uric acid, (3) or the purine bases (4). The possi b i l i t y that urea might be a precursor of uric acid was investigated extensive-l y (3). Urea was injected into animals along with malonic, l a c t i c , pyruvic, propionic, dialuric, glyceric or barbituric acids. In no case was any significant increase i n uric acid production observed. In 1935, Baldwin (3) fed urea along with tartronic acid and obtained an appreci-able increase i n uric acid production. This result was later shown to be not valid since when urea and tartronic acid were injected instead of fed, no increase i n uric acid was observed. Therefore, the results obtained by feeding were attributed to bacterial activity i n the gut. In 1943 Sehoenheimer and Barnes (5), i n experiments using urea labelled with nitrogen^ a s tracer, showed that urea i s not a precursor of poly-nucleotide purines. In 1946, Bloch (6) fed rats urea labelled with nitrogenl5 and found that most of the heavy nitrogen was excreted as urea. A small but relatively insignificant amount was found i n the body protein and urinary ammonia. From his results Bloch concluded that urea i s not a precursor of creatine nor i s i t u t i l i z e d i n purine formation. He stated that the participation of urea i n any intermediary reactions i s not indicated. More recently experiments have been carried out on the metabolism of urea using urea labelled with the long-lived radioactive isotope of carbon. In 1948, Leifer, Roth and Henrpelmann(7) studied the gross met-abolism of urea i n mice after injecting them with carbons-labelled urea. They found that after 48 hours approximately twenty percent of the i n -jected carbon^ appeared i n the expired air as carbon dioxide. The re-maining eighty percent was excreted i n the urine as urea. A very rapid i n i t i a l excretion of radioactivity was observed and the biological half-l i f e of urea was found to be five hours. No explanation was given as to the mechanism of the formation of carbon dioxide from the injected urea. 3. In connection with a research problem involving the biochemistry of the purines and pyrimidines, urea labelled with carbon^ was prepared i n this laboratory. Since labelled urea was readily available i t was deemed of interest to carry out investigations for the purpose of at-tempting to c l a r i f y or supplement present knowledge on the subject of urea metabolism. Labelled urea was injected intraperitoneally into rats and the animals were placed i n a metabolism cage which permitted collection of expired air and excreta. Urine, feces, expired a i r , certain organs, blood and carcass were analyzed quantitatively for radioactivity. Three separate experiments were carried out. In order to perfect the analytical methods a preliminary experiment was performed using urea of low specific activity. Then two experiments were carried out using urea of high spe-c i f i c activity. In the f i r s t of these latter experiments, the animal was kept i n the metabolism cage for 48 hours after injection. It was found that after 48 hours, 94 percent of the injected radioactive carbon had been excreted, either as urea or carbon dioxide. Of the tot a l excreted, 32 percent was found i n the expired a i r and 68 percent i n the urine. Over one-half of the t o t a l was excreted i n the f i r s t six hours after i n -jection. In the third experiment, the animal was k i l l e d after three hours i n order to study the distribution of carbon^ shortly after injection. In a l l three experiments the radioactivity i n the expired a i r reached a maximum i n the second hour indicating rapid metabolism of a significant portion of the injected urea. The fact that the urea excreted i n the urine i n the f i r s t three hours was found to have a very high specific activity showed that some of the injected urea was probably excreted with-out undergoing metabolism. In the three hour experiment, analyses of 4. l i v e r , kidney and blood indicated that most of the radioactivity i n the l i v e r and kidney was present as urea. In the blood, the majority of the radioactivity was present as urea while small amounts were found as carbon dioxide and i n protein. Analysis of urine showed also that the carbon^ was present only as urea. These experiments showed that when isotopic urea was injected into rats, a considerable portion of the urea was metabolised rapidly to carbon dioxide. The metabolism reached a maximum i n the second hour after injection. The i n i t i a l excretion of urea of very high specific activity indicated that some of the urea was excreted without undergoing metabolism. Analysis of l i v e r , kidney and blood after three hours showed that almost a l l of the radioactivity was present as urea and therefore very l i t t l e carbon"^ had been incorporated into tissue at this time. At the end of 48 hours about five percent of the injected carbon^ was retained i n the carcass of the animal and may have been i n -corporated into body protein. Due to the specific activity of the urea available for injection, i t was necessary to inject a f a i r l y large amount of urea, namely 10 mgm. This resulted i n the level of urea i n the animal being above the physio-logical level and therefore the experiments were carried out under ab-normal conditions. In future experiments, i f urea of higher specific activity were synthesized, the same number of counts could be injected with a smaller amount of urea making the conditions of the experiment more nearly physiological. It would be of interest also to carry out continuous injection experiments whereby small amounts of radioactive urea were injected over a period of time to increase the amount of carbonM present without greatly increasing the total amount of urea. 5. Also as a suggestion for further research, since investigations on the metabolism of urea using tracer techniques have involved the use of 15 14 nitrogen J and carbon as tracers i n separate experiments, the idea immediately presents i t s e l f that the subject of urea metabolism might be c l a r i f i e d further by experiments i n which urea, doubly labelled with nitrogen1-* and carbon 1^, was used for injection. It would be interesting too, to inject labelled urea into hepatectomized animals to determine whether the l i v e r i s the site of urea metabolism as well as urea synthesis. 6. EXPERIMENTAL A. Measurement of Radioactivity -A very convenient and efficient method of measuring the radio-activity of materials containing carbon^ i s to oxidize the carbon to carbonate and precipitate the carbonate as the barium or calcium salt for counting. In this investigation the radioactive materials were oxidized by wet oxidation and the carbonate was precipitated as the ba-rium salt. The barium carbonate precipitates were then f i l t e r e d through small, brass Buchner-type f i l t e r funnels as described by Armstrong and Schubert (8). The B-activity of the precipitates i n the dishes was mea-sured by means of an end-window Geiger-Mueller tube. This method was found to be easy of application and yielded reproducible results. It also furnished a gravimetric analysis of the to t a l carbon i n the sample. (i) Oxidation - A l l compounds and tissues were converted to barium carbonate prior to counting, by wet oxidation using the com-bustion mixture described by Van Slyke and Folch (9) and the apparatus as shown i n Figure 1. Weighed samples of the material to be oxidized, containing about 10 mgms. of carbon, were placed i n combustion tube A and 300 mgms. of D Fig. 1. Apparatus used for wet oxidation of organic cprapounds and tissues. 8. pulverized potassium iodate were added. Approximately 25 cc. of carbon dioxide-free sodium hydroxide solution, prepared by the method described by Calvin et a l (10) were placed i n centrifuge tube F. The system was evacuated through E by means of the water pump and the combustion f l u i d , consisting of chromic, phosphoric and fuming sulphuric acids, was added cautiously from D through stop-cock C without breaking the vacuum. The solution was then boiled for several minutes to complete the oxidation. The solution was allowed to cool, a tube containing soda lime was i n -serted i n D, and a i r allowed to enter by opening stop-cock C. This helped to sweep any carbon dioxide l e f t i n the combustion tube over into F. The time required to carry out a combustion was about 20 minutes. To precipitate barium carbonate, the method of Regier (11) was used. Ammonium chloride (1.57 g) was added to reduce the alk a l i n i t y of the sodium hydroxide solution containing absorbed carbon dioxide. The solution was then heated to boiling and excess of a saturated solution of barium chloride was added to precipitate barium carbonate. When cool, the precipitate was f i l t e r e d through the brass f i l t e r funnels, weighed and counted. This method was found to y i e l d a precipitate consisting of large particles of barium carbonate which f i l t e r e d readily, did not crack when dried, and was easily smoothed for counting. To test the method, several combustions were carried out on samples of urea, cholesterol, benzoic acid and sodium carbonate, with results as shown i n Table 1. ( i i ) Counting - Before counting, the barium carbonate precipitates i n the brass counting dishes were smoothed with a flattened glass rod i n order to obtain reproducible counting geometry. The B-activity of the precipitates was then measured by placing the dishes i n a reproducible 9. position beneath a thin end-window, self-quenching Geiger-Mueller tube connected to an electronic "scale of 64". TABLE 1 Recovery of carbon from organic compounds oxidized by wet oxidation. Substance oxidized Sample weight mg. Carbon mg. Found percent Theoretical percent Cholesterol 10.10 8.48 83.98 83.87 10.60 8.85 83o46 10.00 8.34 83.39 Urea 45.60 9.13 20.01 20.00 50.10 10.03 20.01 Benzoic acid 15.0 10.42 69.45 68.85 11.0 7.59 68.98 Sodium carbonate 79.0 8.93 11.31 11.32 82.0 9.20 11.22 The B-particles emitted by carbon ^ are of such low energy (0.154 Mev) that the barium carbonate sample thicknesses represent an appreciable fraction of the mean particle range (10). This results i n the introduction of large errors i n counting due to self-absorption losses. To eliminate the need for self-absorption corrections, samples were made thick enough so that the radiations originating i n the layer farthest removed from the counter were completely absorbed by the inter-vening layers. Such samples are said to be " i n f i n i t e l y thick" samples. Since the activity observed from " i n f i n i t e l y thick" samples i s propor-tional to the specific activity of the sample, no self-absorption cor-rections are necessary. * Nuclear Instrument and Chemical Corporation 10. To obtain the thickness of sample corresponding to "in f i n i t e thickness" for the counting assembly used i n this laboratory, an ac t i -vity saturation curve was plotted as shown i n Figure 2. Portions of a precipitate of radioactive barium carbonate were f i l t e r e d successively through the same brass counting dish yielding samples of increasing thickness but constant area. Each sample was weighed and counted. When the counts no longer increased with increase i n sample thickness, the saturation activity had been reached and the samples were of "i n f i n i t e thickness". The fraction of maximum activity was calculated' for each sample and was then plotted against thickness of sample i n mgms. per cm2. From the curve i t can be seen that no appreciable increase i n activity was observed after the thickness of sample had reached 25 mgms. per cm2. This thickness, therefore, was taken as "in f i n i t e thickness". The area of the brass counting dishes was 5.34 cm2 so that a barium carbonate precipitate weighing 132.7 mgms. or more was " i n f i n i t e l y thick". Sam-ples of less than "in f i n i t e thickness" were corrected by reference to the activity saturation curve. The thickness of the sample i n mgms. per cm was calculated and the fraction of maximum activity was read from the curve. The observed count was then divided by the fraction of maximum activity to yield the actual count. A l l counts were corrected for background, counter performance and coincidence error. Counter performance was corrected by use of a barium carbonate standard. Coincidence error which i s due to the re-solving time of the counting apparatus was corrected by reference to a correction curve for coincidence error (12). Sufficient counts were recorded so that the counting error was i n no case greater than 2.5 percent. 11. Fig. 2 ; Activity Saturation Curve of BaC-^Oj. 12. It has been advocated that a brass collar should be inserted i n the counting dishes before measuring the activity i n order to eliminate high counting rates caused by the small amount of radioactive precipi-tates which sometimes adheres to the top and inner sides of the dishes after f i l t e r i n g . It was found i n this investigation that a film of paraffin placed over the top and inside surface of the dishes after drying and weighing effectively eliminated these superfluous counts. B. \'."'-';.) Synthesis of labelled urea - The method used to prepare labelled urea was that described by Zbarsky and Fischer (13). Dry ammonia gas was passed over radioactive barium carbonate contained i n a porcelain boat i n a combustion furnace maintained at a temperature of 850° C for three hours. The barium cyanamide so formed was converted to cyanamide by addition of sulphuric acid and the cyanamide was hydro-lysed to urea by boiling for ten minutes under reflux with dilute hydro-chloric acid. The following equations represent the reactions. (An asterisk represents radioactive carbon). BaC03 4- 2 NH5 Ba = N -C a N + 3 H20 BaN - t 5 N +- H 2S0 4 — ^ NHg - C - N + BaS04 In a typical experiment 360 mgms. of urea were obtained from 1185 mgms. of barium carbonate. This represents a yi e l d of 89 percent of theoretical. The material melted at 131 - 132° C and a mixture of authentic urea and the synthesized compound also melted at 131 - 132° C. It was possible that some of the radioactivity i n the synthetic urea was present as a radioactive impurity. To determine whether more 13. than one radioactive component was present, the urea was subjected to analysis by paper partition chromatography (7). A water solution of urea was made up containing five mgm. of urea per ml. Portions of this solution containing 50 micrograms of urea were spotted on each of two strips of Whatman No. 1 f i l t e r paper, if- inches i n width. The strips wer,e then placed i n a large glass jar with the tops of the strips im-mersed i n solvent, the jar was sealed and the chromatograms were allowed to run u n t i l the solvent had almost reached the bottom of the strips. The solvents used were butanol saturated with water and a solution of butanol, ethanol, and water, i n equal parts by volume. The f i l t e r paper strips were removed, dried and placed i n contact with Blue Brand X-ray fil m i n the absence of light for 120 hours. After developing, the radio-autographs were found to show only one spot i n each case which indicated there was only one radioactive component present. The Rf values were found to be .531 when using butanol-water as a solvent and .262 when using butanol-ethanol-water. Further proof of the absence of any radioactive impurity i n the synthetic urea was obtained from experiments carried out to determine the radioactivity of the urea solution used for injection. A saline solution of radioactive urea containing 20 mgms. of urea per ml. was prepared to use for injection and for analysis. 0.5 mg. of this solu-tion was made up to 50 ml. i n a volumetric flask. Aliquots of this solution were analysed by wet oxidation as previously described and by hydrolysis with the enzyme urease as follows. One ml. of the urea solu-tion was pipetted into a large test tube and 1 ml. of glycerol-urease, prepared by method of Koch (14), was added along with 1 ml. of phosphate buffer. In every case where required, carrier was added to ensure a thick sample. The solution was incubated for one-half hour at 40° G. After incubation, the tube was connected to another large test tube containing 25 ml. of 10 percent carbonate-free sodium hydroxide solution and to a second tube containing about 10 ml. of the alkaline solution. A few drops of octyl alcohol were added to prevent foaming and the solution was acidified with 3 ml. of concentrated phosphoric acid. Evolved carbon dioxide was then absorbed i n the sodium hydroxide sol-ution by aerating one-half hour. This was effected by drawing carbon dioxide-free a i r through the solution by means of the water pump. Pre-cipitation of barium carbonate and counting was carried out as previously described. Hydrolysis of urea may be represented by the following equa-tion: + ,0 urease ,0 H NH2C'- NH2 > NH40-C^0-NH4 ^ C0 2 + H20 + 2 NH3 H20 The enzyme urease i s noted for i t s sp e c i f i c i t y (15) and w i l l not hydrolyse even substituted ureas. Since recovery of radioactivity by hydrolysis with urease was i n excellent agreement with that obtained by wet oxidation, as shown i n Table 2, this was almost conclusive proof that no radioactive impurity was present i n the synthetic urea. * When determining the radioactivity of solutions i t i s common practice to evaporate the solutions to dryness and oxidize the residue. This evaporation is necessary to prevent a decrease i n strength of the oxidizing solution by dilution with water. Such a procedure was used when analysing solutions of urea for radioactivity. Evaporation at f i r s t was carried out over a boiling water bath but the results obtained were found to be i n poor agreement with those obtained by hydrolysis with urease. Therefore, several wet oxidations were performed on sam-ples of radioactive urea some of which had been evaporated to dryness 15. over a boiling water bath and others at room temperature i n vacuo, over concentrated sulphuric acid. The results were compared with those obtained by hydrolysis with urease as shown i n Table 2. More consistent results were obtained by evaporation i n vacuo, at room temperature. It was decided therefore to use this procedure when determining the radioactivity of solutions of radioactive compounds. TABLE 2 Recovery of radioactivity and barium carbonate from urea after analysis by various methods. Method of analysis Weight of BaC05 obtained Radioactivity observed Evaporation at 100° and wet oxidation tt tt tt grams 146.4 164.5 163.1 161.9 counts per minute 5995 6600 6950 6050 Evaporation i n vacuo and wet oxidation tt tt 165.2 164.5 164.5 6252 6240 6300 Hydrolysis with urease w it 165.5 167.6 165.0 6250 6295 6240 Gs. Metabolism Experiments -(i) Apparatus - In order to collect expired carbon dioxide, urine, and feces of a rat to which labelled urea had been administered, a metabolism apparatus was constructed, modelled after that described by Armstrong, Schubert and Lindenbaum (16). The apparatus i s shown i n Figure 3. 16. Fig. 3. Apparatus for collection of expired COg and excreta of a rat. 17. After the animal had been injected i t was placed i n a wire cage contained i n an inverted, 10 l i t r e glass bottle, A. The bottle was closed by means of a lucite cover and clamped on tigh t l y by means of thumb-screws. A rubber gasket served to make the bottle a i r tight. Joint M was sealed with plasticine. E was connected to the water pump and the water was then turned on which drew air through the apparatus. Carbon dioxide was removed from the intake a i r by passing i t successi-vely through scrubbing towers B and C which contained sodium and barium hydroxide solutions respectively. Tube D contained saturated sodium chloride solution and served to control the humidity of the air. Urine was collected i n tube F and feces at the bottom of the cage, G. Ex-pired carbon dioxide was absorbed i n sodium hydroxide solution contained i n tower H and when desired i t was possible to switch the stream of expired air to tower J by turning stopcock K, closing clamp N and open-ing clamp 0. With such an arrangement it.was possible to collect ex-pired carbon dioxide for any given period without interruption of the experiment or loss of expired a i r . A trap, L containing barium hydrox-ide solution placed between the absorption towers and the water pump .' served to test for completeness of absorption of carbon dioxide in. towers H and J and to prevent escape of radioactive carbon dioxide i n the event of incomplete absorption. The recovery of radioactivity was quantitative as shown i n experiments i n which a known amount of radioactive barium carbonate was placed i n the apparatus and decomposed with acid. The average recovery of radioactivity from three t r i a l s was 98.9 percent of the theoretical. ( i i ) Rat 1 - In order to perfect the analytical methods, a preliminary experiment was performed using urea of low specific 18. activity for injection. A female albino rat weighing 190 grams was given an intraperi-toneal injection of 0.50 ml. of a saline solution of urea containing 500 mgms. of urea per ml. The specific activity was 20 counts per min-ute per mgm. of urea and the total counts were 5000 counts per minute. The rat was placed i n the metabolism apparatus and carbon dioxide free air drawn through. Expired carbon dioxide was collected for hourly periods and urine at two and four hours after injection. Four hours after the injection the rat was removed, anaesthetized with nembutal and i t s spleen, kidney, stomach and intestine were excised. The organs and feces were placed i n weighed beakers and dried to constant weight at 90° C. The organs and feces were then pulverized i n a mortar. Por-tions were weighed out and the carbon present oxidized, precipitated as barium carbonate, and counted. The carcass was dissolved i n hot 20 percent potassium hydroxide solution. When cool, the solution was made up to one l i t r e and aliquot samples were evaporated to dryness. The carbon i n the residues was then oxidized by wet oxidation and precipitated as barium carbonate for counting. The sodium hydroxide solution containing expired carbon dioxide was drained from the tower into a one l i t r e volumetric flask. The towers were washed with carbon dioxide free d i s t i l l e d water and the washings added to the solution i n the volumetric flask. The solution was then made up to one l i t r e . Aliquots containing sufficient car-bonate to yi e l d " i n f i n i t e l y thick" samples were analysed for radio-activity by precipitating the carbonate with barium chloride. The urine was made up to volume i n a volumetric flask and the 19. radioactivity was determined by two methods. F i r s t , the total radio-activity was determined by evaporating aliquots to dryness and oxidiz-ing the residues by wet oxidation. Secondly, the radioactivity present as urea was determined, by hydrolysis with urease as previously des-cribed. The results are shown i n Table 3. TABLE 3 Carbon1^ content of the tissues and excreta of a rat k i l l e d 4 hours after intraperitoneal injection of 250 mg. of Cr^-urea with a spe-c i f i c activity of 20 counts/min./mg. urea. Total counts injected, 5,000 per minute. Material Radioactivity found examined Total counts per minute % of radioacti-vity injected Expired air, 1st hour 220 4.4 " " 2nd hour 320 6.4 11 " 3rd hour 300 6.0 " " 4th hour 210 4.2 Liver, kidney, spleen 950 19.0 Stomach, intestine and feces trace t Urine 2,900 58.0 Total 4,900 98.0 As indicated i n Table 3 of the tot a l carbon^ excreted, 26 percent was excreted i n expired a i r and 74 percent i n urine. The excretion of radioactivity i n expired air and 74 percent i n urine. The excretion of radioactivity i n expired a i r reached a maximum i n 20. the second hour after injection. The counts recorded for l i v e r , spleen, and kidney were only slightly above the background count and therefore, are subject to large errors. Recovery of radioactivity was 98 percent of the total injected. It i s interesting to note that similar results were obtained i n this preliminary experiment using urea of very low specific activity for injection as were obtained by injection of the urea of high specific a c t i v i t y as described i n the following experiments. ( i i i ) Rat 2 - This experiment was carried out i n order to study the excretion of radioactive carbon by a rat which had been injected with radioactive urea of high specific activity and placed i n the metabolism apparatus for a period of 48 hours. A male albino rat weighing 250 grams was given an intraperitoneal injection of 0.5 ml. of a saline solution of urea containing 20 mgms. of urea per ml. The specific activity of the urea was 26,900 counts per minute per mgm. of urea so that a t o t a l count of 269,900 counts per min-ute, were injected. The rat was placed immediately i n the metabolism apparatus and expired carbon dioxide was collected at 1, 2, 3, 6, 12, 30, 36 and 48 hours, after injection and urine at 6, 12, 24, 30, 36 and 48 hours. At the end of the 48 hour period, the animal was removed and an-esthetized with nembutal. Heparin was injected into the jugular vein, the carotid artery was severed, and the blood was collected i n a small beaker. The stomach, kidney, l i v e r , intestine and spleen were then ex-cised. The kidney, l i v e r , spleen, feces, urine, expired air and carcass were analysed for radioactivity i n a manner similar to that described i n the previous experiment. To remove the fat from the stomach and intestine, after drying to constant weight, these tissues were extracted with a solution of alcohol and ether i n a micro-soxhlet apparatus. Only a trace of radioactivity was found i n both the fat and the fat-extracted residue. 21. The blood was centrifuged and the plasma decanted. The red cells were dried, pulverized and counted directly. Plasma was also counted directly after evaporation to dryness. Such an insignificant amount of radioactivity was found i n both red cells and plasma that they were not oxidized for counting as barium carbonate. Results of the analyses on rat 2 are shown i n Tables 4 and 5. TABLE 4 Carbon1^ content of the tissues and excreta of a rat k i l l e d 48 hours after intraperitoneal injection of 10 mgs. of C^-urea with a specific activity of 26,900 counts/min./mg. urea. Total counts injected, 269,000/minute. Material Radioactivity found examined Total counts per minute fo of radioactivity inje cted Expired air 81,300 3 0 . 2 Urine 170,180 6 3 . 2 Liver, kidneys, spleen, stomach, intestines 800 0 . 3 Feces 1 , 0 0 0 0.4 Carcass 13,070 4.8 Total 2 6 6 , 3 5 0 98.9 From Table 4 can be seen that of the total carbon^ injected, approximately five percent remained i n the carcass after 48 hours, indicating a possible fixation of carbon^ i n the body tissue. No significant amount of radioactivity was found i n any of the organs studied. Recovery of radioactivity was 98.9 percent. The feces were 22. found to contain about 1,000 counts per minute but since some con-tamination of the feces with urine occurred, this result has l i t t l e significance. Table 5 shows the amount of radioactivity excreted i n urine and expired a i r at various times after injection. TABLE 5 14 Excretion of carbon i n expired air and urine, by Rat 2 after intra-peritoneal injection of carbon 1 4 as urea (269,000 counts per minute). Period of Radioactivity excreted i n : collection Urine Expired a i r Total Specific counts activity c.p.m. cpm/mg.C Total counts c.p.m. Specific activity cpm/mg.C 0 - 1 hour 10,200 90. 1 - 2 hours 16,700 124. 2 - 3 hours 13,700 78. 3 - 6 hours 19,600 40. 0 - 6 hours (urine only) 92,340 5,660 6 - 1 2 hours 69,560 1,480 - 12,800 9. 12 - 24 hours 4,420 98 4,000 1.4 24 - 30 hours 1,360 40 1,500 1.0 30 - 36 hours 1,775 37 1,100 0.7 36 - 48 hours 730 19 1,700 0.56 Total 170,185 81,300 23. From Table 5 i t can be calculated that, of the total counts excreted 32 percent were excreted i n the expired a i r and 68 percent i n the urine. The excretion of radioactivity i n the expired a i r reached a maximum i n the second- hour after injection as was observed i n the experiments on Rat 1. The large amount of carbon 1 4 and the high spe-c i f i c activity of the urea excreted i n the urine i n the f i r s t 12 hours indicated that a considerable portion of the injected urea was excreted TABLE 6 Results of f i l t e r paper chromatographic analysis of urine and urea using n-butanol-ethanol-water and n-butanol-water as solvents. Material Rf values n-butanol-water ethanol-butanol-water Synthetic urea .262 .531 Urine at 6th hour .264 .55 Urine at 12th hour .269 .54 unchanged. After the 12th hour, the t o t a l counts excreted and specific activity of both the urea and carbon dioxide descreased sharply. It i s interesting to observe that the to t a l counts per minute excreted i n the last 36 hours i n urine and expired a i r were almost identical, being 8,285 and 8,300 respectively. Figure 4 shows the cumulative percentage of carbon 1 4 excreted i n urea and carbon dioxide plotted against time. The time taken for excretion of half of the injected radioactivity was about five hours. This agrees with the results obtained by Leifer, Roth and Hempelmann (7) 24', 100 80 LJ o UJ 60 o x : o _i i o >-! X U_ ! LU O 40 20 0 ® URINE o EXPIRED AIR • % RADIOACTIVITY REMAINING IN ANIMAL -©--o 0 10 20 30 TIME (HOURS) 40 50 Fig. 4. Excretion of C 1^ i n urine and expired a i r by a rat after being injected intraperitoneally with 0.5ml of a solution of radio-active urea containing 20mgm per ml and having a specific activity of 26,900cpm per mgm of urea. 2 5 . who found the biological half-live of urea injected into mice to be five hours. Two descending f i l t e r paper chromatograms were run on the urine using n-butanol saturated with water as solvent i n the f i r s t , and a solution of equal parts by volume of ethanol, n-butanol, and water i n the second. Radioautographs were made of the f i l t e r paper strips and the spots produced were found to have approximately the same Rf values as those obtained with the synthetic radioactive urea, as shown i n Table 6. This showed that no appreciable amount of radioactive com-pounds other than urea were present i n the urine. (iv) Rat 3 - This experiment was carried out to study the distribution of carbon 1 4 from radioactive urea i n a rat shortly after injection of the material. The animal was injected with urea of high specific activity and removed from the metabolism apparatus after three hours. A male albino rat weighing 250 grams was'given an intraperitoneal •injection of 0 . 5 ml. of a saline solution of urea containing 20 mgm.' of urea per ml. The specific a c t i v i t y was 3 1 , 2 5 0 counts per minute per mgm. ,of urea so that a total of 3 1 2 , 5 0 0 counts per minute were injected. • The rat was placed i n the metabolism apparatus and expired a i r was collected for hourly intervals and urine at the end of the three hour period. There were no feces. After three hours the animal was,removed, anesthetized with nembutal and the blood was collected i n a small beaker as described i n the previous experiment. The stomach, intestine, kidney and l i v e r were then excised. Stomach, intestine, expired a i r and carcass were analysed for radioactivity as i n the previous experiments. 26. Liver and kidney were minced i n a Waring Blendor with ice-cold five percent trichloroacetic acid solution to precipitate protein and the mixture was centrifuged. The residues were washed with trichloro-acetic acid and the washings added to the supernatant f l u i d . The t r i -chloroacetic extracts were i n turn extracted with ether to remove the TABLE 7 Carbon 1 4 content of tissues and excreta of a rat k i l l e d 3 hours after intraperitoneal injection of 10 mg. of C^-urea with a specific a c t i -vity of 31,260 counts/mg. urea. Total counts injected, 312,600/minute. Material Radioactivity found examined Total counts per minute % of injected counts Expired air,1st hour 11,060 3.5 " " 2nd hour 11,250 3.6 " " 3rd hour 7,400 2.3 Urine 99,000 31.7 Blood urea 4,300 1.3 Blood C0 2 110 .03 Kidney urea 4,300 1.3 Liver urea 3,300 1.0 Carcass 140,000 35.1 Total 280,720 89.8 trichloroacetic acid and the water solution remaining from this ex-traction was analysed for urea by hydrolysis with urease. The protein residues were dried, pulverised and counted directly. The ether ex-tract was analysed for radioactivity by d i s t i l l i n g off the ether and 27. counting the dried residue, directly. No radioactivity was found i n the ether extract and a small amount was found i n the protein residue. To determine radioactivity present as carbon dioxide, 1 ml. aliquots of blood were pipetted into large test tubes and these were connected to test tubes containing sodium hydroxide solution. The blood was acidified with hydrochloric acid and aeration carried out to enable the evolved carbon dioxide to be absorbed i n the sodium hydroxide solution. From the results shown i n Table 7 i t was calculated the recovery of radioactivity was only 90 percent. This was probably due to the fact that some urine of high specific activity was lost after the animal had been removed from the cage. Also the animal continued to respire while blood was being collected and therefore some carbon"'"4 was undoubtedly lost as carbon dioxide. Only a trace of radioactivity was found i n blood protein, sto-mach, intestine and the fat extracts of stomach and intestine, i n d i -cating that l i t t l e carbon 1 4 had been fixed i n the tissues at this time. Slightly more radioactivity was found i n the kidney than i n the l i v e r which was probably due to the concentrating action of the tubles i n the kidney. Almost a l l of the radioactivity found i n blood, kidney and l i v e r was present as urea. By the method of analysis used i t was not possible to t e l l where or i n what form the large amount of radio-activity (140,000 c.p.m.) found i n the carcass existed but i t very probably was present as unabsorbed urea. The specific activity of the urea excreted was 49,500 counts per minute per mgm. of carbon which suggests that some of the injected urea was excreted unchanged as i n Rat 2. As i n Rats 1 and 2, excretion 28. of carbon 1^ i n the expired a i r reached a maximum i n the second hour after injection. The urine i n addition to analysis by wet oxidationsand urease hydrolysis was analysed for radioactivity present as urea by the xan-thydrol method as described by Allen and Luck (17). Five ml. of urine were pipetted into a large centrifuge tube and 20 mgm. of nonradioactive urea were added to produce an i n f i n i t e l y thick sample. An equal volume of glacial acetic acid was added and then 2 ml. of a 10 percent solution of xanthydrol i n methanol were added dropwl.se. The reagents were mixed intimately by shaking and the dixanthydryl ureide was allowed to pre-cipitate for one hour. The tube was then centrifuged and the preci-pitate washed i n the centrifuge tube with 50 percent acetic acid. The precipitate was f i l t e r e d onto a previously weighed brass counting dish and washed with methyl alcohol. After drying overnight i n vacuo over phosphorus pentoxide the dixanthydryl ureide was weighed and counted. The total counts obtained were 74,000 counts per minute. Yankwich (10) has shown that there i s a difference i n back-scattering of particles between samples of barium carbonate and organic compounds such as xan-thydrol. Using Yankwich's correction factor of 1.28 the t o t a l counts i n urine by xanthydrol method were -94,700 counts per minute which i s i n good agreement with the results obtained by wet oxidation and hydrolysis with urease. This was further proof of the absence of any radioactive compound other than urea i n the urine. 29. DISCUSSION The results of the experiments which have been described show that when carbon 1 4 was administered to rats i n the form of urea, about seventy percent of the isotopic carbon was excreted as urea. A sig-nificant portion, up to 30 percent, was metabolised and appeared in^the expired air as carbon dioxide over a period of 48 hours after administra-tion. A rapid i n i t i a l excretion of carbon 1 4 was observed and the very high specific activity of the urea excreted i n the f i r s t three hours indicated that a portion of the injected radioactive urea was excreted directly without undergoing hydrolysis. The specific activity and to t a l counts excreted i n expired air i n a l l three experiments reached a maxi-mum i n the second hour after injection as shown i n Figure 5 indicating that the injected urea was rapidly metabolised. Very l i t t l e fixation of radioactive carbon i n tissue was ob^ served. Almost a l l of the carbon 1 4 found i n blood, l i v e r and kidney after three hours was present as urea. More radioactivity was found i n kidney than i n l i v e r as would be expected due to the concentrating action of the tubules i n the kidney. Only a trace of radioactivity was found i n the other organs tested and i n blood protein. About 45 percent of the injected carbon 1 4 was found i n the carcass of the animal i n the 5 1 4 3 H 2 I o r <r N 2 ° o L l I X c J ^ Q 5 ' UJ o u. UJ 3 I 0 t= CO > H 1- z O Z> < ° * -I u . u . 4 -3 c o 2 RAT 1 250 MG. C1 4 UREA INJECTED (5000 COUNTS / MIN.) RAT 2 10 MG. C I 4UREA INJECTED (269,000 COUNTS / MIN.) RAT 3 10 MG. C , 4UREA INJECTED (312,600 C0UNTS/MIN.) 0 1 2 3 4 5 6 INTERVAL AFTER INJECTION OF C14UREA (HOURS) Fig. 5. Excretion of i n expired a i r by Rats 1,2,and 3 after intraperitoneal injection of Cl4 as urea. i 31. three hour experiment. Since only a trace, at the most, of radioacti-v i t y i n any form other than urea was found i n any of the organs tested at this time, i t seems very l i k e l y that the carbon 1 4 found i n the car-cass was present as unabsorbed urea and not fixed i n body tissue. In the 48 hour experiment, about five percent of the injected carbon^ 4 was found to be retained i n the carcass of the rat at the end of the 40 hour period. This was believed to have been l a i d down i n body tissue for the following reasons. Half of the injected radioactive carbon was excreted at the end of five hours and after the twelfth hour a sharp decrease i n excretion of carbon 1 4 occurred indicating that the majority of the injected carbon 1 4 had either been excreted directly as urea or metabolised and excreted. Therefore, i t seems very l i k e l y that the carbon 1 4 remaining i n the carcass after as long a period as 48 hours must have been present i n body tissue. This seems, l i k e l y since other experiments (16) have shown that radioactive carbon dioxide administered as labelled bicarbonate i s l a i d down i n various tissues. It should be mentioned here that the poss i b i l i t y exists that the carbon 1 4 found i n expired air 48 hours after injection could have been derived from bac-t e r i a l decomposition of small amounts of urine adhering to the inside of the metabolism cage. From Table 5 i t can be seen that the specific activity of the urea excreted at any time i s much higher than that of the carbon dioxide excreted i n the same period. This disagrees with the results obtained by Mckenzie and duVigneaud (18) who injected methionine labelled i n the methyl group with carbon 1 4 into rats and found that the specific a c t i -vities of the urea excreted and the carbon dioxide exhaled on the same day were equal. Recently, however, Weisburger, Weisburger and Morris (19) 32. administered carbon 1 ^ -labelled 2-acetyl amino fluorene to rats and observed that the specific activity of the urea excreted over an 88 hour period was always much higher than that of the carbon dioxide exhaled i n the same period as was observed i n this investigation. The fact that injected urea instead of being entirely excreted i s hydrolysed i n vivo, as shown by these experiments, indicates that urea may play a part i n intermediary metabolism. Fitzgerald (20) has recently demonstrated the presence of urease activity i n l i v e r , kidney and stomach of various animals including the rat. In the latte r animal, the stomach contains about 10 times as much urease activity as l i v e r or kidney, and this led Fitzgerald to postulate that urea's chief meta-bolic role may be one of protecting the gastric mucosa by supplying ammonia to neutralize hydrochloric acid i n the stomach. Ammonia derived from urea might also be used i n l i v e r or kidney for other reactions such as transamination. This i s an attractive theory since free ammonia is extremely toxic whereas urea is ubiquitous yet innocuous. 33. SUMMARY 1. A method for routine analysis of carbon 1 4 as barium carbonate was established for this laboratory. The thickness of barium carbonate sample corresponding to " i n f i n i t e thickness" was found to be 25 mgm. o per cm. or 132.7 mgm. for the counting dishes used. 2. Labelled urea was synthesized according to method of Zbarsky and Fischer and a y i e l d of 89 percent of the theoretical was obtained. The radioactivity of the synthetic compound was shown to be present only as urea by hydrolysis with the specific enzyme urease. The synthetic urea was also subjected to f i l t e r paper partition chro-matography and radioautographs of the f i l t e r paper strips showed the absence of any detectable radioactive impurity. 3. Apparatus was set up and the method established for the oxidation of the carbon of organic compounds and tissues by wet oxidation using the Van Slyke-Folch oxidizing mixture. 4. The metabolism of urea was studied i n a series of three experiments i n vdiich radioactive urea was injected intraperitoneally into rats. The excreta, expired a i r , certain organs, blood and carcasses of the animals were quantitatively analysed for carbon^content. 34. Approximately 65 percent of the injected carbon 1^ was excreted as urea. A rapid i n i t i a l excretion of carbon 1^ occurred and from the high specific activity of the urea excreted i n the f i r s t three hours after injection a considerable portion of the injected urea must have been excreted unchanged. Approximately 30 percent of the injected carbon^ appeared i n the expired air. Both the specific activity of the carbon dioxide and the total counts excreted reached a maximum i n the second hour after injection. In one experiment i n which the animal was sacrificed three hours after injection, a l l but a trace of the radioactivity present i n kidney, l i v e r and blood was i n the form of urea and no incorporation of carbon^ into tissue was observed. In another experiment i n which the,animal was sacrificed 48 hours after injection the incorporation into body tissue of about five percent of the carbon 1^ injected, was indicated. 35. BIBLIOGRAPHY i 1. Krebs, H.A. and Henseleit, K. - Z. Physiol. Chem., 210, 33 (1932). 2. Beard, H.A. and Pizzalato, P. - J. Biochem. Japan, 28, 421 (1938). 3. Baldwin, E. - Biochem. J., 2£, 1538 (1935). 4. Minkowski, 0. - Arch. Exp. Path, and Pharmacol., 21, 41 (1886). 5. Schoenheimer, R. and Barnes, F.W. - J. Biol. Chem., 151, 123 (1943). 6. Block, K. - J. Biol. Chem., 165, 469 (1946). 7. Leifer, E., Roth, L.J. and Hempelmann, L.H. - Science, 108, 748 (1948). 8. Armstrong, W.D. and Schubert, J. - Anal. Chem., 20_, 270 (1948). 9. Van Slyke, D.D. and Folch, J. - J. Biol. Chem., 136, 509 (1940). 10. Calvin, M., Heidelberger, C , Reid, J.C, Tolbert, B.M. and Yankwich, P.F. - Isotopic carbon, John Wiley and Sons, New York, (1949). 11. Regier, R.B. - Anal. Chem., 21, 1020 (1949). 12. Tracerlog, Tracerlab Inc., Number 3 (1947). 13. Zbarsky, S.H. and Fischer, I. - Can. J. of Res., B 2 7 , 81 (1948). 14. Koch, F.C. - Practical Methods i n Biochem., William Wood and Co., (1934). 15. Baldwin, E. - Dynamic Aspects of Biochemistry, 2nd edition, Cambridge (1948). 16. Armstrong, W.D., Schubert, J. and Lindenbaum, A. - Proc. Soc. Exp. Biol, and Med., 68, 233 (1948). 17. Allen, J.M. and Luck, H. - J. Biol. Chem., 82, 695 (1929). 36. 18. Mckenzie, C.G., and du Vigneaud, . - J. Biol. Chem., 172, 353 (1948). 19. Weisburger, J.H., Weisburger, E.K. and Morris, H.P. - J. Nat. Can. Inst., 11, 797 (1951). 20. Fitzgerald, 0. - Biochem. J., £7 , J.X (1950). i i 

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