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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 CARBON  14  - 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 t h i s t h e s i s as conforming to the standard required from candidates f o r the degree of MASTER OF ARTS  Members of the Department of  THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1952  ABSTRACT Urea, l a b e l l e d with carbon*-^  w a  s  synthesized and  administered 14  to r a t s by i n t r a p e r i t o n e a l i n j e c t i o n .  The excretion of carbon 1  was  followed by analysing the urine, feces and expired a i r q u a n t i t a t i v e l y f o r r a d i o a c t i v i t y at various times a f t e r i n j e c t i o n , and the d i s t r i b u t i o n of the isotope was studied by analysis of organs, blood and carcass.  A portion of the i n j e c t e d urea was r a p i d l y metabolised,  approxi-  mately 30 percent of the isotope being excreted i n the expired a i r a f t e r 12 hours.  The highest output of C^^Og  after injection.  occurred during the second hour  The majority of the remaining isotope was excreted i n  the urine as urea.  A f t e r three hours only a small percentage of the  i n j e c t e d c a r b o n ^ was present i n the kidney, l i v e r , and blood l a r g e l y i n the form of urea.  A f t e r 48 hours, approximately  f i v e percent of  the c a r b o n ^ i n j e c t e d as urea remained i n the carcass and appeared to be f i x e d i n the t i s s u e s .  ACKNOWLEDGMENTS This research was carried out under a grant from the National Research Council.  The author i s indebted to Dr. S.H. Zbarsky f o r h i s encouragement, c r i t i c i s m and advice throughout the course of t h i s research.  TABLE OF CONTENTS  INTRODUCTION  . .  1  EXPERIMENTAL A.  Measurement of Radioactivity (i) (ii)  Oxidation  6  Counting  8  B.  Synthesis of Labelled Urea  C.  Metabolism Experiments (i)  DISCUSSION  12  Apparatus  15  (ii)  Rat 1  17  (iii)  Rat 2  20  (iv)  Rat 3  . . .  v  25  29  SUMMARY  33  BIBLIOGRAPHY  35  INTRODUCTION The compound urea i s the p r i n c i p a l end-product of nitrogen metabolism i n mammals.  The synthesis of urea has been shown t o 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 a f t e r hepatectomy.  In  v i t r o studies using tissue s l i c e s under nearly p h y s i o l o g i c a l conditions have also shown that only l i v e r t i s s u e i s capable of synthesizing urea. The actual mechanism of urea formation i s not d e f i n i t e l y 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 f a t , carbohydrate or non-nitrogenous protein residues combine with ornithine t o y i e l d c i t r u l l i n e .  C i t r u l l i n e i s then  converted i n t o arginine by addition of another molecule of ammonia and arginine through the influence of the enzyme arginase breaks down t o y i 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 a f t e r i t had been formed.  I t 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) u r i c acid, (3) or the purine bases (4). The p o s s i b i l i t y that urea might be a precursor of u r i c a c i d was investigated extensivel y (3).  Urea was i n j e c t e d into animals along with malonic, l a c t i c ,  pyruvic, propionic, d i a l u r i c , g l y c e r i c or b a r b i t u r i c acids.  In no case  was any s i g n i f i c a n t increase i n u r i c acid production observed.  In 1935,  Baldwin ( 3 ) fed urea along with t a r t r o n i c acid and obtained an appreciable increase i n u r i c a c i d production.  This r e s u l t was l a t e r shown t o  be not v a l i d since when urea and t a r t r o n i c a c i d were i n j e c t e d instead of fed, no increase i n u r i c acid was observed.  Therefore, the r e s u l t s  obtained by feeding were attributed to b a c t e r i a l a c t i v i t y i n the gut. In 1943 Sehoenheimer and Barnes (5), i n experiments using urea l a b e l l e d with n i t r o g e n ^  a s  tracer, showed that urea i s not a precursor of poly-  nucleotide purines.  In 1946,  Bloch (6) fed rats urea l a b e l l e d with  nitrogenl5 and found that most of the heavy nitrogen was excreted as urea.  A small but r e l a t i v e l y i n s i g n i f i c a n t amount was found i n the body  protein and urinary ammonia.  From h i s r e s u l t s 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 p a r t i c i p a t i o n 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 l a b e l l e d with the long-lived radioactive isotope of carbon.  In 1948, L e i f e r , Roth and Henrpelmann(7) studied the gross met-  abolism of urea i n mice a f t e r i n j e c t i n g them with c a r b o n s - l a b e l l e d urea. They found that a f t e r 48 hours approximately twenty percent of the i n jected c a r b o n ^ appeared i n the expired a i r as carbon dioxide. maining eighty percent was excreted i n the urine as urea.  The r e -  A very rapid  i n i t i a l excretion of r a d i o a c t i v i t y was observed and the b i o l o g i c a l h a l f l i f e of urea was found to be f i v e 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 l a b e l l e d with c a r b o n ^ was prepared i n t h i s laboratory.  Since l a b e l l e d urea was r e a d i l y available i t was  deemed of interest to carry out investigations f o r the purpose of a t tempting t o c l a r i f y or supplement present knowledge on the subject of urea metabolism.  Labelled urea was injected i n t r a p e r i t o n e a l l y i n t o r a t s and the animals were placed i n a metabolism cage which permitted c o l l e c t i o n of expired a i r and excreta.  Urine, feces, expired a i r , c e r t a i n organs,  blood and carcass were analyzed quantitatively f o r r a d i o a c t i v i t y . separate experiments were c a r r i e d out.  Three  In order to perfect the a n a l y t i c a l  methods a preliminary experiment was performed using urea of low s p e c i f i c activity.  Then two experiments were c a r r i e d out using urea of high spe-  cific activity.  In the f i r s t of these l a t t e r experiments, the animal was  kept i n the metabolism cage f o r 48 hours a f t e r i n j e c t i o n .  I t was found  that a f t e r 48 hours, 94 percent of the injected radioactive carbon had been excreted, either as urea or carbon dioxide.  Of the t o t 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 s i x hours a f t e r i n jection.  I n the t h i r d experiment, the animal was k i l l e d a f t e r three hours  i n order t o study the d i s t r i b u t i o n of c a r b o n ^ shortly a f t e r i n j e c t i o n . In a l l three experiments the r a d i o a c t i v i t y i n the expired a i r reached a maximum i n the second hour i n d i c a t i n g r a p i d metabolism of a s i g n i f i c a n t p o r t i o n of the i n j e c t e d urea.  The f a c t 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 s p e c i f i c a c t i v i t y showed that some of the injected urea was probably excreted without undergoing metabolism.  In the three hour experiment, analyses of  4. l i v e r , kidney and blood indicated that most of the r a d i o a c t i v i t y i n the l i v e r and kidney was present as urea.  In the blood, the majority of  the r a d i o a c t i v i t y was present as urea while small amounts were found as carbon dioxide and i n protein.  Analysis of urine showed also that the  c a r b o n ^ was present only as urea. These experiments showed that when i s o t o p i c urea was  injected  into r a t s , a considerable portion of the urea was metabolised r a p i d l y to carbon dioxide. after injection.  The metabolism reached a maximum i n the second hour The i n i t i a l excretion of urea of very high s p e c i f i c  a c t i v i t y indicated that some of the urea was excreted without undergoing metabolism.  Analysis of l i v e r , kidney and blood a f t e r three hours  showed that almost a l l of the r a d i o a c t i v i t y was present as urea and therefore very l i t t l e carbon"^ had been incorporated i n t o t i s s u e at t h i s time.  At the end of 48 hours about f i v e percent of the i n j e c t e d  c a r b o n ^ was retained i n the carcass of the animal and may have been i n corporated i n t o body protein.  Due to the s p e c i f i c a c t i v i t y of the urea available f o r i n j e c t i o n , i t was necessary t o i n j e c t a f a i r l y large amount of urea, namely 10  mgm.  This resulted i n the l e v e l of urea i n the animal being above the physiol o g i c a l l e v e l and therefore the experiments were carried out under abnormal conditions.  In future experiments, i f urea of higher s p e c i f i c  a c t i v i t y were synthesized, the same number of counts could be injected with a smaller amount of urea making the conditions of the experiment more nearly p h y s i o l o g i c a l .  I t would be of interest also t o carry out  continuous i n j e c t i o n experiments whereby small amounts of radioactive urea were i n j e c t e d over a period of time to increase the amount of carbonM present without greatly increasing the t o t a l amount of urea.  5. Also as a suggestion f o r f u r t h e r research, since investigations on the metabolism of urea using t r a c e r techniques have involved the use of 15 nitrogen  J  14 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 l a b e l l e d with nitrogen -* and carbon ^, was used f o r i n j e c t i o n . 1  1  I t would be i n t e r e s t i n g  too, to i n j e c t l a b e l l e d urea i n t o hepatectomized animals to determine whether the l i v e r i s the s i t e of urea metabolism as well as urea synthesis.  6.  EXPERIMENTAL A.  Measurement of Radioactivity -  A very convenient and e f f i c i e n t method of measuring the radioa c t i v i t y of materials containing c a r b o n ^ i s to oxidize the carbon t o carbonate and p r e c i p i t a t e the carbonate as the barium or calcium s a l t f o r counting.  In t h i s i n v e s t i g a t i o n the radioactive materials were  oxidized by wet oxidation and the carbonate was p r e c i p i t a t e d as the barium s a l t .  The barium carbonate p r e c i p i t a t e s 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 - a c t i v i t y of the p r e c i p i t a t e s i n the dishes was mea-  sured by means of an end-window Geiger-Mueller  tube.  This method was  found t o be easy of a p p l i c a t i o n and y i e l d e d reproducible r e s u l t s . I t also furnished a gravimetric analysis of the t o t a l carbon i n the sample.  (i)  Oxidation  -  A l l compounds and tissues were converted t o  barium carbonate p r i o r t o counting, by wet oxidation using the combustion mixture described by Van Slyke and Folch (9) and the apparatus as shown i n Figure 1. Weighed samples of the material t o be oxidized, containing about 10 mgms. of carbon, were placed i n combustion tube A and 300 mgms. of  D  F i g . 1. Apparatus used f o r wet oxidation of organic cprapounds and t i s s u e s .  8. pulverized potassium iodate were added.  Approximately 25 cc. of carbon  dioxide-free sodium hydroxide solution, prepared by the method described by C a l v i n 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  s o l u t i o n was then boiled f o r several minutes to complete the oxidation. The s o l u t i o n was allowed t o 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 i n t o F.  The time required t o carry out a combustion was about 20 minutes. To p r e c i p i t a t e barium carbonate, the method of Regier (11) was  used.  Ammonium chloride (1.57 g) was added to reduce the a l k a l i n i t y of  the sodium hydroxide s o l u t i o n containing absorbed carbon dioxide.  The  s o l u t i o n was then heated to b o i l i n g and excess of a saturated s o l u t i o n of barium chloride was added to p r e c i p i t a t e barium carbonate.  When cool,  the p r e c i p i t a t e 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 p r e c i p i t a t e consisting of  large p a r t i c l e s of barium carbonate which f i l t e r e d r e a d i l y , d i d not crack when dried, and was e a s i l y smoothed f o r counting.  To test the method, several combustions were c a r r i e d out on samples of urea, cholesterol, benzoic acid and sodium carbonate, with r e s u l t s as shown i n Table 1. (ii)  Counting - Before counting, the barium carbonate p r e c i p i t a t e s  i n the brass counting dishes were smoothed with a f l a t t e n e d glass rod i n order to obtain reproducible counting geometry.  The B - a c t i v i t y of the  p r e c i p i t a t e s was then measured by placing the dishes i n a reproducible  9. p o s i t i o n beneath a t h i n end-window, self-quenching  Geiger-Mueller tube  connected t o an electronic "scale of 64".  TABLE 1 Recovery of carbon from organic compounds oxidized by wet oxidation.  Substance oxidized  Sample weight  Carbon  mg. mg.  Found percent  Theoretical percent  Cholesterol  10.10 10.60 10.00  8.48 8.85 8.34  83.98 83o46 83.39  83.87  Urea  45.60 50.10  9.13 10.03  20.01 20.01  20.00  Benzoic acid  15.0 11.0  10.42 7.59  69.45 68.98  68.85  Sodium carbonate  79.0 82.0  8.93 9.20  11.31 11.22  11.32  The B-particles emitted by carbon ^ are of such low energy (0.154 Me ) that the barium carbonate sample thicknesses represent an v  appreciable  f r a c t i o n of the mean p a r t i c l e range (10). This r e s u l t s i n  the introduction of large errors i n counting due t o self-absorption losses.  To eliminate the need f o r self-absorption corrections, samples  were made t h i c k enough so that the radiations o r i g i n a t i n g i n the layer farthest removed from the counter were completely absorbed by the i n t e r vening layers.  Such samples are said to be " i n f i n i t e l y t h i c k " samples.  Since the a c t i v i t y observed from " i n f i n i t e l y t h i c k " samples i s proport i o n a l t o the s p e c i f i c a c t i v i t y of the sample, no self-absorption corrections are necessary.  *  Nuclear Instrument and Chemical Corporation  10. To obtain the thickness of sample corresponding t o " i n f i n i t e thickness" f o r the counting assembly used i n t h i s laboratory, an a c t i v i t y saturation curve was p l o t t e d as shown i n Figure 2. Portions of a p r e c i p i t a t e of radioactive barium carbonate were f i l t e r e d successively through the same brass counting d i s h y i e l d i n g 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 a c t i v i t y had been reached and the samples were of " i n f i n i t e thickness".  The f r a c t i o n of maximum a c t i v i t y was calculated' f o r each  sample and was then p l o t t e d against thickness of sample i n mgms. per cm . 2  From the curve i t can be seen that no appreciable increase i n a c t i v i t y was observed a f t e r the thickness of sample had reached 25 mgms. per cm . 2  This thickness, therefore, was taken as " i n f i n i t e thickness".  The area  of the brass counting dishes was 5.34 cm so that a barium carbonate 2  p r e c i p i t a t e weighing 132.7 mgms. or more was " i n f i n i t e l y t h i c k " .  Sam-  ples of l e s s than " i n f i n i t e thickness" were corrected by reference t o the a c t i v i t y saturation curve. per cm  The thickness o f the sample i n mgms.  was calculated and the f r a c t i o n of maximum a c t i v i t y was read  from the curve.  The observed count was then divided by the f r a c t i o n  of maximum a c t i v i t y t o y i e l d the actual count.  A l l counts were corrected f o r 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 r e -  solving time of the counting apparatus was corrected by reference to a correction curve f o r coincidence error (12).  S u f f i c i e n t counts were  recorded so that the counting error was i n no case greater than percent.  2.5  11.  F i g . 2 ; A c t i v i t y Saturation Curve of BaC-^Oj.  12. It has been advocated that a brass c o l l a r should be inserted i n the counting dishes before measuring the a c t i v i t y i n order t o eliminate high counting rates caused by the small amount of radioactive p r e c i p i tates which sometimes adheres to the top and inner sides of the dishes after f i l t e r i n g .  I t was found i n t h i s i n v e s t i g a t i o n that a f i l m of  p a r a f f i n placed over the top and inside surface of the dishes a f t e r drying and weighing e f f e c t i v e l y eliminated these superfluous counts.  B.  \'."'-';.) Synthesis of l a b e l l e d urea - The method used t o prepare  l a b e l l e d urea was that described by Zbarsky and Fischer (13). Dry ammonia gas was passed over radioactive barium carbonate contained i n a p o r c e l a i n boat i n a combustion furnace maintained at a temperature of 850° C f o r three hours.  The barium cyanamide so formed was converted  to cyanamide by addition of sulphuric acid and the cyanamide was hydrolysed to urea by b o i l i n g f o r ten minutes under r e f l u x with d i l u t e hydrochloric acid.  The following equations represent the reactions. (An  asterisk represents radioactive carbon). BaC0 4- 2 NH 3  Ba = N -C  a  5  N+ 3 H 0 2  BaN - t 5 N +- H S0 — ^ NHg - C - N + BaS0 2  4  4  In a t y p i c a l experiment 360 mgms. of urea were obtained from 1185 mgms. of barium carbonate. of t h e o r e t i c a l .  This represents a y i e l d of 89 percent  The material melted at 131 - 132° C and a mixture o f  authentic urea and the synthesized compound also melted at 131 - 132° C.  It was possible that some of the r a d i o a c t i v i t y 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 t o analysis by paper p a r t i t i o n chromatography (7). A water s o l u t i o n of urea was made up containing f i v e mgm. of urea per ml. Portions of t h i s s o l u t i o n containing 50 micrograms of urea were spotted on each o f two s t r i p s of Whatman No. 1 f i l t e r paper, if- inches i n width.  The s t r i p s  wer,e then placed i n a large glass j a r with the tops of the s t r i p s immersed i n solvent, the j a r was sealed and the chromatograms were allowed to run u n t i l the solvent had almost reached the bottom of the s t r i p s . The solvents used were butanol saturated with water and a s o l u t i o n of butanol, ethanol, and water, i n equal parts by volume.  The f i l t e r paper  s t r i p s were removed, d r i e d and placed i n contact with Blue Brand X-ray f i l m i n the absence of l i g h t f o r 120 hours.  A f t e r 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 t o 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 c a r r i e d out t o determine the r a d i o a c t i v i t y of the urea s o l u t i o n used f o r i n j e c t i o n .  A saline  s o l u t i o n of radioactive urea containing 20 mgms. of urea per ml. was prepared to use f o r i n j e c t i o n and f o r analysis.  0.5 mg. of t h i s solu-  t i o n was made up t o 50 ml. i n a volumetric f l a s k .  Aliquots of t h i s  solution were analysed by wet oxidation as previously described and by hydrolysis with the enzyme urease as follows.  One ml. of the urea solu-  t i o n was pipetted i n t o 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, c a r r i e r was added t o ensure a  t h i c k sample.  The s o l u t i o n was incubated f o r one-half hour at 40° G.  A f t e r incubation, the tube was connected to another large t e s t tube containing 25 ml. of 10 percent carbonate-free sodium hydroxide solution and to a second tube containing about 10 ml. of the a l k a l i n e s o l u t i o n . A few drops of o c t y l alcohol were added to prevent foaming and the s o l u t i o n was a c i d i f i e d with 3 ml. of concentrated phosphoric acid. Evolved carbon dioxide was then absorbed i n the sodium hydroxide s o l u t i o n 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-  c i p i t a t i o n of barium carbonate and counting was carried out as previously described.  Hydrolysis of urea may be represented by the following equa-  tion:  +  ,0 NH C'- NH 2  2  urease > H0  ,0 NH 0-C^0-NH 4  4  H ^ C0 + H 0 + 2 NH3 2  2  2  The enzyme urease i s noted f o r i t s s p e c i f i c i t y (15) and w i l l not hydrolyse even substituted ureas.  Since recovery o f r a d i o a c t i v i t y by  hydrolysis with urease was i n excellent agreement with that obtained by wet oxidation, as shown i n Table 2, t h i s was almost conclusive proof that no radioactive impurity was present i n the synthetic urea. *  When determining the r a d i o a c t i v i t y of solutions i t i s common  practice t o evaporate the solutions to dryness and oxidize the residue. This evaporation i s necessary t o prevent a decrease i n strength of the oxidizing s o l u t i o n by d i l u t i o n with water.  Such a procedure was used  when analysing solutions of urea f o r r a d i o a c t i v i t y .  Evaporation at  f i r s t was c a r r i e d out over a b o i l i n g water bath but the r e s u l t s 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 b o i l i n g water bath and others at room temperature over concentrated sulphuric a c i d .  i n vacuo,  The r e s u l t s were compared with  those obtained by hydrolysis with urease as shown i n Table 2.  More  consistent r e s u l t s were obtained by evaporation i n vacuo, at room temperature.  I t was decided therefore t o use t h i s procedure when  determining the r a d i o a c t i v i t y of solutions of radioactive compounds.  TABLE 2 Recovery of r a d i o a c t i v i t y and barium carbonate from urea a f t e r analysis by various methods.  Method of analysis  Weight of BaC0 obtained grams  Evaporation at 100° and wet oxidation  tt tt tt  Evaporation i n vacuo and wet oxidation  tt tt  Hydrolysis with urease  w it  Gs.  5  Radioactivity observed counts per minute  146.4 164.5 163.1 161.9  5995 6600 6950 6050  165.2 164.5 164.5  6252 6240 6300  165.5 167.6 165.0  6250 6295 6240  Metabolism Experiments -  (i)  Apparatus -  In order to c o l l e c t expired carbon dioxide,  urine, and feces of a r a t to which l a b e l l e d urea had been administered, a metabolism apparatus was constructed, modelled a f t e r that described by Armstrong, Schubert and Lindenbaum (16). Figure 3.  The apparatus i s shown i n  16.  F i g . 3. Apparatus f o r c o l l e c t i o n of expired COg and excreta of a r a t .  17. A f t e r 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 b o t t l e , A.  The b o t t l e  was closed by means of a l u c i t e cover and clamped on t i g h t l y by means of thumb-screws.  A rubber gasket served to make the b o t t l e a i r t i g h t .  Joint M was sealed with p l a s t i c i n e .  E was connected to the water pump  and the water was then turned on which drew a i r through the apparatus. Carbon dioxide was removed from the intake a i r by passing i t successively through scrubbing towers B and C which contained sodium and barium hydroxide solutions respectively.  Tube D contained saturated sodium  chloride solution and served t o control the humidity of the a i r . was c o l l e c t e d i n tube F and feces at the bottom of the cage, G.  Urine Ex-  pired carbon dioxide was absorbed i n sodium hydroxide s o l u t i o n contained i n tower H and when desired i t was possible to switch the stream of expired a i r to tower J by turning stopcock K, closing clamp N and opening clamp 0.  With such an arrangement it.was possible t o c o l l e c t ex-  p i r e d carbon dioxide f o r any given period without i n t e r r u p t i o n of the experiment or loss of expired a i r .  A trap, L containing barium hydrox-  ide s o l u t i o n placed between the absorption towers and the water pump .' served to test f o r completeness of absorption of carbon dioxide i n . towers H and J and to prevent escape of radioactive carbon dioxide i n the event of incomplete absorption.  The recovery of r a d i o a c t i v i t y 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 r a d i o a c t i v i t y from three t r i a l s was 98.9 percent of the theoretical.  (ii)  Rat 1 - In order to perfect the a n a l y t i c a l methods, a  preliminary experiment was performed using urea of low s p e c i f i c  18. activity f o r injection.  A female albino r a t weighing 190 grams was given an i n t r a p e r i toneal i n j e c t i o n of 0.50 ml. of a saline s o l u t i o n of urea containing 500 mgms. of urea per ml. ute  The s p e c i f i c a c t i v i t y was 20 counts per min-  per mgm. of urea and the t o t a l counts were 5000 counts per minute.  The r a t was placed i n the metabolism apparatus and carbon dioxide free a i r drawn through.  Expired carbon dioxide was c o l l e c t e d f o r hourly  periods and urine at two and four hours a f t e r i n j e c t i o n .  Four hours  a f t e r the i n j e c t i o n the r a t was removed, anaesthetized with nembutal and i t s spleen, kidney, stomach and i n t e s t i n e were excised.  The organs  and feces were placed i n weighed beakers and d r i e d 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, p r e c i p i t a t e d as barium carbonate, and counted.  The carcass was dissolved i n hot 20 percent potassium hydroxide solution.  When cool, the s o l u t i o n was made up to one l i t r e and aliquot  samples were evaporated t o dryness.  The carbon i n the residues was  then oxidized by wet oxidation and p r e c i p i t a t e d as barium carbonate f o r counting.  The sodium hydroxide solution containing expired carbon dioxide was drained from the tower i n t o a one l i t r e volumetric f l a s k .  The  towers were washed with carbon dioxide free d i s t i l l e d water and the washings added to the s o l u t i o n i n the volumetric f l a s k . was then made up to one l i t r e .  The s o l u t i o n  Aliquots containing s u f f i c i e n t car-  bonate to y i e l d " i n f i n i t e l y t h i c k " samples were analysed f o r radioa c t i v i t y by p r e c i p i t a t i n g the carbonate with barium chloride.  The urine was made up to volume i n a volumetric f l a s k and the  19. r a d i o a c t i v i t y was determined by two methods.  F i r s t , the t o t a l r a d i o -  a c t i v i t y was determined by evaporating aliquots to dryness and o x i d i z ing the residues by wet oxidation.  Secondly, the r a d i o a c t i v i t y present  as urea was determined, by hydrolysis with urease as previously described.  The r e s u l t s are shown i n Table 3. TABLE 3  Carbon ^ content of the tissues and excreta of a r a t k i l l e d 4 hours a f t e r intraperitoneal i n j e c t i o n of 250 mg. of Cr^-urea with a spec i f i c a c t i v i t y of 20 counts/min./mg. urea. Total counts injected, 5,000 per minute. 1  Radioactivity found Material examined % of r a d i o a c t i v i t y injected  Total counts per minute Expired a i r , 1st hour "  220  4.4  "  2nd hour  320  6.4  11  "  3rd hour  300  6.0  "  "  4th hour  210  4.2  L i v e r , kidney, spleen  950  19.0 t  Stomach, i n t e s t i n e and feces  trace  Urine  2,900  58.0  Total  4,900  98.0  As indicated i n Table 3 of the t o t a l c a r b o n ^ excreted, 26 percent was excreted i n expired a i r and 74 percent i n urine.  The  excretion of r a d i o a c t i v i t y i n expired a i r and 74 percent i n urine. The excretion of r a d i o a c t i v i t y i n expired a i r reached a maximum i n  20. the second hour a f t e r i n j e c t i o n .  The counts recorded f o r l i v e r , spleen,  and kidney were only s l i g h t l y above the background count and therefore, are subject to large errors. of the t o t a l injected.  Recovery of r a d i o a c t i v i t y was 98 percent  It i s i n t e r e s t i n g t o note that s i m i l a r r e s u l t s  were obtained i n t h i s preliminary experiment using urea of very low s p e c i f i c a c t i v i t y f o r i n j e c t i o n as were obtained by i n j e c t i o n of the urea of high s p e c i f i c a c t i v i t y as described i n the following experiments. (iii)  Rat 2 -  This experiment was carried out i n order to study  the excretion of radioactive carbon by a r a t which had been i n j e c t e d with radioactive urea of high s p e c i f i c a c t i v i t y and placed i n the metabolism apparatus f o r a period of 48 hours.  A male albino rat weighing 250 grams was given an intraperitoneal i n j e c t i o n of 0.5 ml. of a saline s o l u t i o n of urea containing 20 mgms. of urea per ml.  The s p e c i f i c a c t i v i t y 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 i n j e c t e d .  The r a t was placed immediately i n the metabolism  apparatus and expired carbon dioxide was c o l l e c t e d at 1, 2, 3, 6, 12, 30, 36 and 48 hours, a f t e r i n j e c t i o n 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 i n j e c t e d into the jugular vein,  the c a r o t i d artery was severed, and the blood was c o l l e c t e d i n a small beaker.  The stomach, kidney, l i v e r , i n t e s t i n e and spleen were then ex-  cised.  The kidney, l i v e r , spleen, feces, urine, expired a i r and carcass  were analysed f o r r a d i o a c t i v i t y i n a manner s i m i l a r t o that described i n the previous experiment.  To remove the f a t from the stomach and i n t e s t i n e ,  a f t e r drying to constant weight, these t i s s u e s were extracted with a s o l u t i o n of alcohol and ether i n a micro-soxhlet apparatus.  Only a trace  of r a d i o a c t i v i t y was found i n both the f a t and the fat-extracted residue.  21. The blood was centrifuged and the plasma decanted. c e l l s were dried, pulverized  and counted d i r e c t l y .  counted d i r e c t l y a f t e r evaporation to dryness.  The red  Plasma was  also  Such an i n s i g n i f i c a n t  amount of r a d i o a c t i v i t y was found i n both red c e l l s and plasma that they were not oxidized f o r counting as barium carbonate.  Results of the analyses on r a t 2 are shown i n Tables 4 and 5. TABLE 4 Carbon ^ content of the tissues and excreta of a rat k i l l e d 48 hours after intraperitoneal i n j e c t i o n of 10 mgs. of C^-urea with a s p e c i f i c a c t i v i t y of 26,900 counts/min./mg. urea. Total counts injected, 269,000/minute. 1  Radioactivity  found  Material examined Total counts per minute Expired a i r Urine Liver, kidneys, spleen, stomach, intestines Feces Carcass  Total  fo of r a d i o a c t i v i t y inje cted  81,300  30.2  170,180  63.2  800  0.3  1,000  0.4  13,070  4.8  266,350  98.9  From Table 4 can be seen that of the t o t a l c a r b o n ^  injected,  approximately f i v e percent remained i n the carcass a f t e r 48 hours, i n d i c a t i n g a possible f i x a t i o n of c a r b o n ^ i n the body t i s s u e .  No  s i g n i f i c a n t amount of r a d i o a c t i v i t y was found i n any of the organs studied.  Recovery of r a d i o a c t i v i t y was 98.9 percent.  The feces were  22. found t o contain about 1,000 counts per minute but since some contamination of the feces with urine occurred, t h i s r e s u l t has l i t t l e significance. Table 5 shows the amount of r a d i o a c t i v i t y excreted i n urine and expired a i r at various times a f t e r i n j e c t i o n . TABLE 5 14 Excretion of carbon i n expired a i r and urine, by Rat 2 a f t e r i n t r a peritoneal i n j e c t i o n of c a r b o n as urea (269,000 counts per minute). 14  Radioactivity excreted i n : Period of collection Urine  Expired a i r  Total Specific counts a c t i v i t y c.p.m. cpm/mg.C  0-1  Total counts c.p.m.  hour  Specific activity cpm/mg.C  10,200  90.  1 - 2 hours  16,700  124.  2-3  hours  13,700  78.  3-6  hours  19,600  40.  12,800  9.  0 - 6 hours (urine only)  92,340  5,660  6-12  hours  69,560  1,480  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 t o t a l counts excreted 32 percent were excreted i n the expired a i r and 68 percent i n the  urine.  The excretion of r a d i o a c t i v i t y i n the expired a i r reached  a maximum i n the second- hour after i n j e c t i o n as was observed i n the experiments on Rat 1.  The large amount of c a r b o n  14  and the high spe-  c i f i c a c t i v i t y 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 i n j e c t e d 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.  Rf values Material n-butanol-water  ethanol-butanolwater  Synthetic urea  .262  .531  Urine at 6th hour  .264  .55  Urine at 12th hour  .269  .54  unchanged.  A f t e r the 12th hour, the t o t a l counts excreted and s p e c i f i c  a c t i v i t y of both the urea and carbon dioxide descreased sharply.  It i s  i n t e r e s t i n g t o observe that the t o t a l counts per minute excreted i n the l a s t 36 hours i n urine and expired a i r were almost i d e n t i c a l , being 8,285 and 8,300 respectively.  Figure 4 shows the cumulative percentage of c a r b o n i n urea and carbon dioxide plotted against time.  14  excreted  The time taken f o r  excretion of half of the i n j e c t e d r a d i o a c t i v i t y was about f i v e hours. This agrees with the r e s u l t s obtained by L e i f e r , Roth and Hempelmann (7)  24',  ® URINE o EXPIRED AIR • % RADIOACTIVITY REMAINING IN ANIMAL  100  80 LJ  o  UJ  -©-  60  o x :o  _i  40 -o  i o >- 20 ! X U_ ! LU O  0 0  10  20 30 TIME (HOURS)  40  50  F i g . 4. Excretion of C ^ i n urine and expired a i r by a rat a f t e r being injected i n t r a p e r i t o n e a l l y with 0.5ml of a solution of radioactive urea containing 20mgm per ml and having a s p e c i f i c a c t i v i t y of 26,900cpm per mgm of urea. 1  25.  who found the b i o l o g i c a l h a l f - l i v e of urea injected i n t o mice t o be f i v e 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 s o l u t i o n 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 s t r i p s 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 c a r r i e d out t o study the  d i s t r i b u t i o n of c a r b o n  14  from radioactive urea i n a r a t shortly a f t e r  i n j e c t i o n of the material.  The animal was i n j e c t e d with urea of high  s p e c i f i c a c t i v i t y and removed from the metabolism apparatus a f t e r three hours. A male albino rat weighing 250 grams was'given an intraperitoneal •injection of 0 . 5 ml. of a saline s o l u t i o n of urea containing 20 mgm.' urea per ml.  The s p e c i f i c a c t i v i t y was  ,of urea so that a t o t a l of rat  312,500  31,250  counts per minute per  of mgm.  counts per minute were i n j e c t e d . • The  was placed i n the metabolism apparatus and expired a i r was c o l l e c t e d  f o r hourly i n t e r v a l s and urine at the end of the three hour period. were no feces.  There  A f t e r three hours the animal was,removed, anesthetized  with nembutal and the blood was c o l l e c t e d i n a small beaker as described i n the previous experiment. were then excised.  The stomach, i n t e s t i n e , kidney and l i v e r  Stomach, i n t e s t i n e , expired a i r and carcass were  analysed f o r r a d i o a c t i v i t y as i n the previous experiments.  26. L i v e r and kidney were minced i n a Waring Blendor with i c e - c o l d f i v e percent t r i c h l o r o a c e t i c acid solution t o p r e c i p i t a t e protein and the mixture was centrifuged.  The residues were washed with t r i c h l o r o -  acetic acid and the washings added t o the supernatant f l u i d . chloroacetic extracts were i n turn extracted  The t r i -  with ether t o remove the  TABLE 7 C a r b o n content of tissues and excreta of a r a t k i l l e d 3 hours a f t e r i n t r a p e r i t o n e a l i n j e c t i o n of 10 mg. of C^-urea with a s p e c i f i c a c t i v i t y of 31,260 counts/mg. urea. Total counts injected, 312,600/minute. 14  Radioactivity  found  Material examined Total counts per minute  Expired a i r , 1 s t hour  % of injected counts  11,060  3.5  "  "  2nd hour  11,250  3.6  "  "  3rd hour  7,400  2.3  99,000  31.7  4,300  1.3  Urine Blood urea Blood C 0  110  2  .03  Kidney urea  4,300  1.3  L i v e r urea  3,300  1.0  Carcass  140,000  35.1  Total  280,720  89.8  t r i c h l o r o a c e t i c acid and the water s o l u t i o n remaining from t h i s ext r a c t i o n was analysed f o r urea by hydrolysis with urease. residues were dried, pulverised  and counted d i r e c t l y .  The p r o t e i n  The ether ex-  t r a c t was analysed f o r r a d i o a c t i v i t y by d i s t i l l i n g o f f the ether and  27. counting the dried residue, d i r e c t l y .  No r a d i o a c t i v i t y was found i n  the ether extract and a small amount was found i n the p r o t e i n residue. To determine r a d i o a c t i v i t y 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 s o l u t i o n .  The  blood was a c i d i f i e d with hydrochloric acid and aeration carried out to enable the evolved carbon dioxide to be absorbed i n the sodium hydroxide solution.  From the r e s u l t s shown i n Table 7 i t was calculated the recovery of r a d i o a c t i v i t y was only 90 percent.  This was probably due to the f a c t  that some urine of high s p e c i f i c a c t i v i t y was l o s t a f t e r the animal had been removed from the cage.  Also the animal continued to respire while  blood was being c o l l e c t e d and therefore some carbon"'" was 4  undoubtedly  l o s t as carbon dioxide.  Only a trace of r a d i o a c t i v i t y was found i n blood p r o t e i n , stomach, i n t e s t i n e and the f a t extracts of stomach and i n t e s t i n e , i n d i cating that l i t t l e c a r b o n  14  had been f i x e d i n the tissues at t h i s time.  S l i g h t l y more r a d i o a c t i v i t y was found i n the kidney than i n the l i v e r which was probably due t o the concentrating a c t i o n of the tubles i n the kidney.  Almost a l l of the r a d i o a c t i v i t y 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 radioa c t i v i t y (140,000 c.p.m.) found i n the carcass existed but i t very probably was present as unabsorbed urea. The s p e c i f i c a c t i v i t y of the urea excreted was 49,500 counts per minute per mgm.  of carbon which suggests that some of the i n j e c t e d  urea was excreted unchanged as i n Rat 2.  As i n Rats 1 and 2, excretion  28. of carbon ^ i n the expired a i r reached a maximum i n the second hour 1  after i n j e c t i o n .  The urine i n addition to analysis by wet oxidationsand urease hydrolysis was analysed f o r r a d i o a c t i v i t y present as urea by the xanthydrol method as described by A l l e n and Luck (17).  Five ml. of urine  were pipetted into a large centrifuge tube and 20 mgm. urea were added t o produce an i n f i n i t e l y thick sample.  of nonradioactive An equal volume  of g l a c i a l acetic acid was added and then 2 ml. of a 10 percent s o l u t i o n of xanthydrol i n methanol were added dropwl.se.  The reagents were mixed  intimately by shaking and the dixanthydryl ureide was allowed t o prec i p i t a t e f o r one hour.  The tube was then centrifuged and the p r e c i -  p i t a t e washed i n the centrifuge tube with 50 percent acetic acid.  The  p r e c i p i t a t e was f i l t e r e d onto a previously weighed brass counting dish and washed with methyl alcohol.  A f t e r drying overnight i n vacuo over  phosphorus pentoxide the dixanthydryl ureide was weighed and The t o t a l counts obtained were 74,000 counts per minute. has shown that there i s a difference i n back-scattering of  counted.  Yankwich (10) particles  between samples of barium carbonate and organic compounds such as xanthydrol.  Using Yankwich's correction f a c t o r 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 r e s u l t s obtained by wet oxidation and hydrolysis with urease.  This was f u r t h e r proof of the absence of any radioactive  compound other than urea i n the urine.  29.  DISCUSSION  The r e s u l t s of the experiments which have been described show that when c a r b o n  14  was administered t o r a t s i n the form of urea, about  seventy percent of the i s o t o p i c carbon was excreted as urea.  A sig-  n i f i c a n t portion, up t o 30 percent, was metabolised and appeared in^the expired a i r as carbon dioxide over a period of 48 hours a f t e r administration.  A rapid i n i t i a l excretion of c a r b o n  14  was observed and the very  high s p e c i f i c a c t i v i t y of the urea excreted i n the f i r s t three hours indicated that a portion of the i n j e c t e d radioactive urea was excreted d i r e c t l y without undergoing hydrolysis.  The s p e c i f i c a c t i v i t y and t o t a l  counts excreted i n expired a i r i n a l l three experiments reached a maximum i n the second hour a f t e r i n j e c t i o n as shown i n Figure 5 i n d i c a t i n g that the i n j e c t e d urea was r a p i d l y metabolised.  Very l i t t l e f i x a t i o n of radioactive carbon i n tissue was ob^ served.  Almost a l l of the c a r b o n  14  found i n blood, l i v e r and kidney  a f t e r three hours was present as urea.  More r a d i o a c t i v i t y was found i n  kidney than i n l i v e r as would be expected due t o the concentrating action of the tubules i n the kidney.  Only a trace of r a d i o a c t i v i t y was found  i n the other organs tested and i n blood protein. the injected c a r b o n  14  About 45 percent of  was found i n the carcass of the animal i n the  5 1  4 3H  RAT 1  250 MG. C UREA INJECTED (5000 COUNTS / MIN.) 14  2 I  or  <r 2 ° N  ^  X Q  u.  UJ  t= > 1O  CO H z Z>  0  °  * -I  LlI  UJ  <  o  5  c  J  '  3 I  u. u. 4 3  co  RAT 2 10 MG. C UREA INJECTED (269,000 COUNTS / MIN.) I4  o  -  RAT 3 10 MG. C UREA INJECTED (312,600 C0UNTS/MIN.) ,4  2  0 1 2 3 4 5 6 INTERVAL AFTER INJECTION OF C UREA (HOURS) 14  F i g . 5. Excretion of i n expired a i r by Rats 1,2,and 3 a f t e r intraperitoneal i n j e c t i o n of Cl4 as urea.  i 31. three hour experiment.  Since only a trace, at the most, of r a d i o a c t i -  v i t y i n any form other than urea was found i n any of the organs t e s t e d at t h i s time, i t seems very l i k e l y that the c a r b o n  14  found i n the car-  cass was present as unabsorbed urea and not f i x e d i n body t i s s u e . In the 48 hour experiment, about f i v e percent of the i n j e c t e d carbon^  4  was found to be retained i n the carcass of the r a t at the end  of the 40 hour period.  This was believed to have been l a i d down i n  body tissue f o r the following reasons.  Half of the i n j e c t e d r a d i o a c t i v e  carbon was excreted at the end of f i v e hours and a f t e r the twelfth hour a sharp decrease i n excretion of c a r b o n majority of the injected c a r b o n  14  urea or metabolised and excreted. the c a r b o n  14  14  occurred i n d i c a t i n g that the  had e i t h e r been excreted d i r e c t l y as Therefore, i t seems very l i k e l y that  remaining i n the carcass a f t e r as long a period as 48 hours  must have been present i n body t i s s u e .  This seems, l i k e l y since other  experiments (16) have shown that radioactive carbon dioxide administered as l a b e l l e d bicarbonate i s l a i d down i n various t i s s u e s .  I t should be  mentioned here that the p o s s i b i l i t y exists that the c a r b o n  14  found i n  expired a i r 48 hours a f t e r i n j e c t i o n could have been derived from bact e r i a l decomposition of small amounts of urine adhering to the i n s i d e of the metabolism cage.  From Table 5 i t can be seen that the s p e c i f i c a c t i v i t y 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 r e s u l t s obtained  by Mckenzie and duVigneaud (18) who i n j e c t e d methionine methyl group with c a r b o n  14  l a b e l l e d i n the  into rats and found that the s p e c i f i c a c t i -  v i t i e s 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 ^ - l a b e l l e d 2-acetyl amino fluorene to r a t s and 1  observed that the s p e c i f i c a c t i v i t y 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 t h i s investigation.  The f a c t that injected urea instead of being e n t i r e l y excreted i s hydrolysed i n vivo, as shown by these experiments, indicates that urea may play a part i n intermediary metabolism.  F i t z g e r a l d (20) has  recently demonstrated the presence of urease a c t i v i t y i n l i v e r , kidney and stomach of various animals including the r a t .  I n the l a t t e r animal,  the stomach contains about 10 times as much urease a c t i v i t y as l i v e r or kidney, and t h i s l e d F i t z g e r a l d to postulate that urea's chief metab o l i c role may be one of protecting the gastric mucosa by supplying ammonia to neutralize hydrochloric a c i d i n the stomach.  Ammonia derived  from urea might also be used i n l i v e r or kidney f o r other reactions such as transamination.  This i s an a t t r a c t i v e theory since free ammonia i s  extremely t o x i c whereas urea i s ubiquitous yet innocuous.  33.  SUMMARY 1.  A method f o r routine analysis of c a r b o n established f o r t h i s laboratory.  14  as barium carbonate was  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. 2.  or 132.7 mgm. f o r the counting dishes used.  Labelled urea was synthesized according to method of Zbarsky and Fischer and a y i e l d of 89 percent of the t h e o r e t i c a l was obtained. The r a d i o a c t i v i t y of the synthetic compound was shown to be present only as urea by hydrolysis with the s p e c i f i c enzyme urease.  The  synthetic urea was also subjected t o f i l t e r paper p a r t i t i o n chromatography and radioautographs of the f i l t e r paper s t r i p s showed the absence of any detectable radioactive impurity. 3.  Apparatus was set up and the method established f o r 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 i n t r a p e r i t o n e a l l y into r a t s . The excreta, expired a i r ,  c e r t a i n organs, blood and carcasses of  the animals were quantitatively analysed f o r carbon^content.  34. Approximately 65 percent of the injected carbon ^ was excreted as 1  urea.  A rapid i n i t i a l excretion of carbon ^ occurred and from the 1  high s p e c i f i c a c t i v i t y of the urea excreted i n the f i r s t three hours a f t e r i n j e c t i o n a considerable portion of the i n j e c t e d urea must have been excreted unchanged.  Approximately 30 percent of the injected c a r b o n ^ appeared i n the expired a i r .  Both the s p e c i f i c a c t i v i t y of the carbon dioxide and  the t o t a l counts excreted reached a maximum i n the second hour a f t e r injection.  In one experiment i n which the animal was s a c r i f i c e d three hours a f t e r i n j e c t i o n , a l l but a trace of the r a d i o a c t i v i t y present i n kidney, l i v e r and blood was i n the form of urea and no incorporation of c a r b o n ^ i n t o tissue was  observed.  In another experiment i n which the,animal was s a c r i f i c e d 48 hours a f t e r i n j e c t i o n the incorporation i n t o body tissue of about f i v e percent of the carbon ^ injected, was indicated. 1  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 . B i o l . Chem., 151, 123 (1943).  6.  Block, K. - J . B i o l . Chem., 165, 469 (1946).  7.  L e i f e r , 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. 10.  Van Slyke, D.D. and Folch, J. - J . B i o l . Chem., 136, 509 (1940). 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. - P r a c t i c a l Methods i n Biochem., William Wood and Co., (1934).  15.  Baldwin, E. - Dynamic Aspects of Biochemistry, 2nd e d i t i o n , Cambridge (1948).  16.  Armstrong, W.D., Schubert, J. and Lindenbaum, A. - Proc. Soc. Exp. B i o l , and Med., 68, 233 (1948).  17.  A l l e n , J.M. and Luck, H. - J. B i o l . Chem., 82, 695 (1929).  36. 18.  Mckenzie, C.G., and du Vigneaud,  19.  Weisburger, J.H., Weisburger, E.K. and Morris, H.P. - J. Nat. Can. Inst., 11, 797 (1951).  20.  i  i  . - J. B i o l . Chem., 172, 353 (1948).  F i t z g e r a l d , 0. - Biochem. J . , £ 7 , J.X (1950).  

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