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Urinary metabolites of S[35]-BAL in the rat Matheson, Alastair Taylor 1953

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URINARY METABOLITES OF S^-BAL IN THE RAT  by ALASTATR TAILOR MATHESON  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Biochemistry  We accept this thesis as conforming to the standard required from candidates for the degree of MASTER OF SCIENCE.  Members of the Department of Biochemistry THE UNIVERSITY OF BRITISH COLUMBIA September, 1953  ABSTRACT A. method for the synthesis of s55_BAL has been described. s35. - iphate s  a  was reduced to the sulphide and converted to Nas55H.  The NaS-^H was then reacted with 2 i3-dibromopropanol to form S^5_ BAiL. The product was  characterized by sulphydryl content, preparation of  two c r y s t a l l i n e d i t h i o l a n s , sulphur analysis and chromatographic behavior.  The metabolism of S^5_BAL was  isotopic BAL was  studied i n the Wistar r a t .  The  administered 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 and the  content:.: of the post-inject ion urine was studied.  S^  The maximum rate of  S^5 excretion i n the urine was observed i n the f i r s t 6 hours after i n j e c t i o n and was followed by a rapid decrease i n S ^ was true f o r both neutral,  excretion.  sulphur and inorganic  This  sulphate.  The  amount of neutral sulphur excreted i n the urine also reached a maximum i n the f i r s t 6'hours and returned to normal within 12 hours.  The  ex-  c r e t i o n of inorganic sulphate, however, remained abnormally high throughout the experiment. was  Approximately 4 - 13% of the  excreted  i n the form of inorganic sulphate while l e s s than 0,5% was  i n the ethereal sulphate.  present  Six possible metabolic products were de-  tected by radiochromatographic studies of the p o s t - i n j e c t i o n urine. These compounds were found to have the following  values when the  chromatograms were run i n a t e r t i a r y butanol-water solvent (70/35): Compound 1  0.07 -  0.10  Compound 2  0.25 -  0.30  Compound 3  0.45 - 0.50  Compound 4  0.60 - 0.65  Compound 5  0.78 - 0.85  Compound 6  0.95 - 0.98  Compound 1 was characterized as"inorganic sulphate while compound 5 was found to be a thiol compound which arose following acid hydrolysis of compound 3. Extraction studies showed only compound 2 to be extracted with ether while a l l but compounds 1 were found to be soluble i n n-butanol.  Compound 4, the major metabolite, was shown to be only  slightly soluble i n n-butanol and insoluble i n ether.  No glucuronide  of BAL or it's metabolites have been found i n the urine and no increase i n glucuronic acid excretion was observed following BAL injection.  The  presence of a large amount of glucose i n the post-injection urine was indicated by chromatographic studies.  ACKNOWLEDGEMENTS The author wishes to thank the Defence Research Board for personal assistance i n the form of a Research Assistantship. The author also wishes to express his sincere thanks to Dr. S.H. Zbarsky for the encouragement and advice offered during the course of this project. This research was supported by a grant from the Defence Research Board.  TABLE OF CONTENTS Page INTRODUCTION  •  . .. .  Properties and uses of BAL . . . . . . . . . . . .  1 5  EXPERIMENTAL A. METHODS I.  II.  Radioactive Measurements a)  Precipitation of S  b)  Measurement of S^5  14  . . . .  17 20  a)  Preparation of S -BAL  . . . .  b)  Characterization of S -BAL . . . . . . .  55  55  iii)  2-phenyl-4-hydroxymethyl-l:3-dithiolan.  24  Chromatography  25  Total. sulphur  26 •  .  •  •  •  •  •  .  .  .  .  o  .  o  o  .  .  .  • • . o « .  Inorganic sulphate  Chromatography .  •  •  •  o  • • . . • < • • • •  a) Detection of BAL and i t s metabolites by means of colour reactions . . . . . . . • b)  24 24  b) Total sulphate c)  20  2 j2-dimethyl-4-hydroxymethyl--l :3-dithiolan • • • • « • • • • • • • • • • .  Urinary Sulphur Analysis . . . . . . . . . . . a)  IV.  .  55  ii)  i  ...........  5 5  Synthesis of S -BAL  i)  III.  14  Detection of BAL and i t s metabolites by means of radioactivity . . . . . . . . . .  26 26 29 30"  30 34  Page ,B» METABOLISM EXPERIMENTS 1,  II.  Experiment  . • • • • « • « « . . " . « « . . .  35  S t a b i l i t y of BAL i n urine . . . . . . . . . .  35  Experiment 2 a)  Nitroprusside t e s t  b)  Chromatographic studies  . . • .  .  37  .  •  38  . . • • .  39  i)  Colour reactions . . . . . . . . . . . .  39  ii)  Radiochromatography . . . . . . . . . . .  41  c)  III.  1  Sulphur analysis  • • • •  i)  T o t a l sulphur . . . . . . .  ii)  T o t a l sulphate . . c . • .  Experiment a)  3  .....  48  •  48  • • • • • • • • • • • • • • • • •  •  Chromatographic studies . . . . . . . . . . i) ii)  iii) iv)  Colour reactions  b)  . . . . . . . . . . e s  56  Radiochromatography  58  Urinary e x t r a c t i o n studies . . . . . . .  62  $ -glucuronidase  ....  .  65  •  66  C h a r a c t e r i z a t i o n of compound 1 as SO^  .  69  Sulphur a n a l y s i s . . . . . . . . . . . . . i) ii)  iii)  55  55  ' v) A c i d h y d r o l y s i s . . . • * . . . . . . vi)  48  T o t a l sulphur . . . . . .  ......  71 .  71  Inorganic sulphate . . . . . . . . . . .  71  Total sulphate . . . . . .  71  ^  Page DISCUSSION  79  . .  SUMMARY BIBLIOGRAPHY  88 .  92  TABLES • . .. • I. II,  ...  Page  Gravimetric determination =of sulphur i n cystine • . 15 Self-absorption of I -particles by benzidine 3  sulphate .samples . , « « « « .o .» , • • • « • « • « , III, IV, V,  Reduction of BaSO^ using carbon black (Darco) . • • 21 Reduction of BaSO^ using sugar charcoal Recovery of added total sulphate  VII, VIII, jX.  21  "\in the determination of 23  e  •5c  VI.  —  Recovery of added S 0^ i n the determination of inorganic sulphate , Ascending chromatography of BAL using various solvent mixtures  29 , '33  Rate of oxidation of BAL i n acidified and neutral u urine • • • « • , , • • * . 35 Radioactive metabolites of BAL present i n the urine of groups A and B . , , . «  ••••••  45  X. Detection of BAL on f i l t e r paper chromatograms , , XI,  XIII, XIV, XV,  47  Total sulphur and total S  XII,  19  35_  sulphur i n urine of groups A and B . , , , , , , , , , , , , , , , , , Total sulphate and total sulphate i n urine of groups A and B . . . . . . . . . . . . . Residual sulphur and residual S35_ iphur i n urine of groups A and B , « , , « . * . . Radioactive metabolites of BAL present i n the urine of groups C and D , . . . . . . , . , , „ , , „ , stl  Total sulphur and total s35_ iphur i n urine of groups C and D , , . . o e , « « « , « « o » » »  49 52 53 61  su  a  72  XVI,  Inorganic sulphate and inorganic -sulphate i n urine of groups C and •«•«•••••'•.  75  XVII,  Total sulphate and total S^-sulphate i n urine of groups C and D , •• • . •  77  Page Ratio of s35-sulphate/neutral-S^-sulphur a f t e r S -BAL i n j e c t i o n . . . . . . .  XVIII.  i n urine 81  35  FIGURES  1.  Apparatus f o r the c o l l e c t i o n of benzidine sulphate . . .  16  2.  Self-absorption c o r r e c t i o n curve f o r S ^  18  3.  Rate of oxidation of BAL. i n neutral urine and i n a c i d i f i e d urine . . .  . . . . . . . .  . . . . . . . . . . .  36  4.  Radiochromatograms of urine from group A .  . . . . . . .  42  5.  Radiochromatograms of urine from group B . . . . . . . . .  43  6.  Radiochromatograms of urine containing added S35_BAL . .  46  7.  Rate of excretion of t o t a l sulphur and t o t a l S35-sulphur at various time i n t e r v a l s . Experiment 2 Excretion of S^5 i n urine of r a t s i n j e c t e d i n t r a p e r i t o n e a l l y with S -BAL. Experiment 2  8.  55  9.  50 51  Rate of excretion of t o t a l sulphate and t o t a l sulphate at various time i n t e r v a l s .  Experiment 2 . .  .  54  10.  Radiochromatograms of urine from group C . . . . . . . .  59  11.  Radiochromatograms of urine from group D . . . . . . < ,  60  12.  Extraction studies on urine from group D .  13.  0  '  0  . . . . . « .  -glucuronidase a c t i o n on group D urine sample . . .  14.  Acid hydrolysis of group D urine .sample . . . . . . .  15.  Characterization of compound 1 as S ^ O ^  16.  Rate of excretion of t o t a l sulphur and t o t a l at various time i n t e r v a l s . Experiment 3  -  64 67  •  . . . . . . . . -sulphur . 7  68 70  3  Page 17.  Excretion of i n the urine of rats injected with S-^-BAL. Experiment 3 . . . • « » < > • • • • • .  18.  Rate of excretion of inorganic sulphate and inorganic s35-sulphate. Experiment 3 . • • • • • »<>  •  76  Rate of excretion of total sulphate and total sulphate. Experiment 3 . . . . . . . . . . . . . . .  »  78  19.  74  INTRODUCTION With the outbreak of World War II the danger arose that lewisite might be used as an agent i n gas warfare.  It was realized that the  existing methods for treatment of lewisite poisoning were unsatisfactory. For this reason, work was started at Oxford i n an attempt to find a compound that would relieve the toxic manifestations of arsenic i n the body. As early as 1909, Ehrlich (1) suggested that the toxic effect of arsenic i n the body was due to i t s reaction with receptor groups i n the protoplasm of the c e l l .  These receptors were thought to be either  hydroxyl or sulphydryl groups.  In 1920, Voegtlin and co-workers (2)  showed that i n order for arsenic to exert i t s toxic effect on the c e l l i t must be present i n the trivalent form as the arsenoxide (R - As = 0). In the study of the trypanocidal action i n rats these workers showed that after injection of arsenoxide there was an immediate f a l l i n the number of parasites i n the blood.  I f , however, glutathione or a similar  sulphydryl compound was injected with the arsenoxide the toxic effect of the arsenoxide could be inhibited. Voegtlin postulated, therefore, that the toxic action of arsenoxide on trypanosomes was due to the blocking of essential sulphydryl groups i n the c e l l .  Other workers were also able to show evidence of a reaction between arsenic and cellular sulphydryl groups (3).  If arsenoxide was  added to a protein solution containing free t h i o l groups, the free t h i o l groups disappeared and the arsenic was found to be firmly bound to the protein.  I f , instead, a protein solution containing no free t h i o l groups  was used no reaction took place and the added arsenoxide could be recovered from the n l t r a f i l t r a t e . In addition to this biochemical evidence, there i s considerable evidence of a chemical nature that arsenicals can react with sulphydryl compounds. Cohen and co-workers (4) were able to show the formation i n vitro of a dithioarsinite when a mercaptan reacted with arsenoxide. S -  R - As = 0  +  2R-L-SH  > R - As'  A  S  arsenoxide  *1  —  .+  HgO  RQ^  dithioarsinite  It was suspected that such a reaction might take place i n vivo. The next step was to determine the type of compound attacked i n the c e l l .  It had been known for many years that arsenic was an i n -  hibitor of certain cellular enzymes. Peters and co-workers (5) at Oxford studied a number of enzyme systems i n order to determine their relative sensitivity to inhibition by arsenicals.  Of these the pyruvate oxidase  system proved to be by f a r the most sensitive.  These workers were also  able to show i n vivo an increase i n the blood pyruvate level at an early stage of lewisite poisoning ( 6 ) .  It was concluded, therefore, that the  inhibition of this enzyme system represented one of the early and out-  standing actions of arsenic i n the tissues (7). When i t was shown (8,9) that the pyruvate oxidase system contained a t h i o l protein essential for enzymatic activity, i t was f e l t that the inhibitory action of arsenicals on this system was due to the effect of arsenic on the essential t h i o l group. However, glutathione and similar monothiols were found to be unable to reverse the effect of lewisite on the pyruvate oxidase system.  Therefore, further work on the nature of the  reaction between lewisite and protein t h i o l groups was carried out.  Using  the derived protein kerateine, Stocken and Thompson (10) were able to show that lewisite combined with the t h i o l groups of this; protein i n the ratio of 1 As : 2 SH. dithiol theory.  These workers postulated what i s now called the  In this theory they proposed that the arsenic had com-  bined with two closely adjacent t h i o l groups to form a ring compound. If, therefore, the special toxicity of lewisite on the pyruvate oxidase system was due to the formation of a ring compound, the only way the toxicity could be overcome would be by introducing into the system a dithiol capable of forming a more stable ring compound with the arsenical. In recent months, the isolation of a cyclic disulphide, °^-lipoic acid, as a component of the pyruvate oxidase system, indicates the validity of the assumptions made by the Oxford group. c< - l i p o i c acid possesses the following structure CH  2  (11);  - (CH ) - CH - (CH )5_ - COOH 2  n  S On reduction of the - S - S  2  S  n  where n = 1, 2 or 3. - bond two -SH groups are formed. These  - 4groups could then combine with lewisite to form a stable ring and thereby disrupt the normal oxidation-reduction function of this compound resulting i n the inhibition of this enzyme system. In order to test the dithiol theory, a number of 1,2- and 1,3dithiols were tested for their antidotal action and were found to be highly effective i n reversing the toxic effects of lewisite poisoning (12). The most effective and the least toxic of these dithiols proved to be 2,3dimercaptopropanol or British Anti-Lewisite (BAL), as i t was called by the Americans.  In vitro BAL reacts with lewisite to form the stable dithio-  arsinite. CH SH  I  2  C 1  CHSH I CHgOH  +  BAL  GH S\  \  j  2  As - CH = CH - CI — > CHS Cl' '\ CHgOH lewisite  As - CH = CH - CI + 2 HCl  dithioarsinite  Whether this reaction takes place i n vivo has never been shown, although indirect evidence indicates that such might be the case.  If the d i -  thioarsinite i s administered subcutaneously to rats i t i s considerably less toxic than are equivalent amounts of lewisite (13).  Furthermore, compara-  tively large amounts of arsenic are excreted i n the urine after injection of the dithioarsinite, a phenomenon which i s also observed when rats, which have been dosed with lewisite, are treated with BAL.  Peters (14) has  summarized the toxic effect of lewisite and the reversal of this effect' by BAL as followsj  - 5SH  Protein  SH  +  S P r o t e i n As 7  Protein /  "\  CH = CH - CI  N  SH  ClgAsCH = CH - CI  BAL  +  SH  As - CH = CH - CI  CHS^  I  CHgOH Properties and Uses of BAL The aliphatic dithiols are usually prepared by the reaction of the corresponding halogen compound with sodium or ammonium hydrosulphide. The reaction i s carried out i n an alcoholic solution and a closed system is employed i n order to prevent gross contamination of the product with complex sulphides (15). The Oxford group prepared BAL by the following sequence of reactions: CH--  CH Br  CHgSH  2  CH  Br,  CH 0H 2  CHBr  +  2 NaSH  —> CHSH  CH 0H 2  +  2 NaBr  I CHgOH  a l l y l alcohol  dibromohydrin  BAL  BAL i s a colourless, oily liquid with a characteristic odour, of a mercaptan. The pure compound boils at 110° C at a pressure of 3 mm., 89° at 0.5 mm. and 78° at 0.1 mm. of mercury.  The compound i s soluble i n  organic solvents but i s only slightly soluble i n water. An aqueous, acidic solution of BAL i s stable at room temperature.  However, i t becomes un-  stable when neutralized to pH 7.0 due to oxidation of the t h i o l groups (16).  If  - 6Chemically, BAL i s slightly acidic.  It forms insoluble mer-  captides with some metal salts such as lead and mercury. With other metal salts i t forms compounds which exhibit intense colors.  The color  reaction with cobalt has been used f o r the microdetermination of BAL (17), although the validity of the results obtained by this method has since been questioned (16). BAL also reacts with aldehydes and ketones to form crystalline compounds which have proven to be useful f o r characterization purposes (15). BAL reacts with acetone as follows:  CRvjSH  CHjyS \ ^ ^ CH^ + 0 = c'  CHSH CH OH  3  >  CHS  ^CHj  + H0 2  CH GH  2  2  The compound formed i s 2:2-dimethyl-4-hydroxymethyl-l:3-dithiolan. Pharmacologically, BAL has been found to produce toxic effects. It has an L D ^ Q f o r rats of 120 mgm./kgm. body weight (16) and several reasons have been suggested to explain this toxicity.  BAL i s a strong  reducing compound and, therefore, interferes with -SH equilibria. It possesses a E ^" of -0.150 V at pH 7.0 (18), and Barron has postulated 0  that BAL, "due to i t s large negative oxidation-reduction potential, would produce enzyme inhibition by maintaining i n the reduced state the electron transfer catalysts of the oxidation enzyme system".  Webb and  Van Heynengen (19) have shown that BAL has a strong inhibiting action on metal enzymes due to the combination of BAL with these metal prosthetic groups.  Although BAL was designed s p e c i f i c a l l y as an agent to reverse the toxic e f f e c t s of l e w i s i t e , i t has since found wide chemotherapeutic use i n other f i e l d s of medicine.  BAL  has proven to be e f f e c t i v e i n reversing  a r s e n i c a l d e r m i t i t i s which often developed during arseno-therapy (20). It has also proven successful i n t r e a t i n g many cases of heavy metal poisoning.  Cases of mercury (21), gold (22), and antimony (14) poisonings have  a l l responded t o BAL treatment.  However, BAL has proven to be a rather  dangerous medicament i n lead poisoning.  I t causes the transfer of lead  from blood ^ahd some tissues to other tissues such as those of the nervous system ( 2 3 ) .  Recent investigationsby Hursh (24) as to the possible use  of BAL i n removing isotopic elements such as polonium from the body have shown that i t accelerates the excretion of polonium and d i v e r t s i t from the hemopoietic organs to the muscles.  Much interest has been stimulated by recent work with BAL i n the neurological f i e l d .  Cumings (25) has reported an increased content of  copper i n the l i v e r and b r a i n tissues during hepatolenticular degeneration. By using BAL to remove t h i s excess copper, S t r e i f f e r and Feldman (26) have found " s t r i k i n g c l i n i c a l improvement i n advanced hepatolenticular degeneration, which was p a r a l l e l e d by a remarkable diminution i n e l e c t r o encephalographic abnormalities".  The use of BAL as an anticarcinogen has also been suggested by Crabtree (27).  BAL was shown to i n h i b i t the carcinogenic action of 3f4-  benzpyrene i n mice.  I t was also shown that 3 s4-benzpyrene could engage  i n d i r e c t chemical action with t h i o l compounds.  The anticarcinogenic  action of BAL, therefore, may be explained as follows.  If one assumes  that the benzpyrene reacts with a t h i o l compound i n the c e l l , the addition of BAL causes the reversal of this reaction and benzpyrene i s tied up by the BAL.  It i s of interest to note that no monothiol i s capable of re-  versing this effect, suggesting that the enzyme tied up by the benzpyrene may-be of the dithiol type. It i s obvious, therefore, from the foregoing examples that BAL has found wide use as a therapeutic agent. However, very l i t t l e work has been done i n the study of the metabolism of thisccompound, either per se or i n the presence of toxic agents. been identified.  None of i t s metabolites have  There is also no evidence of the formation i n vivo  of a cyclic compound with lewisite. It is f e l t , therefore, that more knowledge of the metabolism of BAL i s a prerequisite to an understanding of the manner i n which BAL acts as an antidote. Much of our present knowledge of the metabolism of BAL i s based on the work of Spray and co-workers at Oxford, and Young and co-workers at Toronto.  Stocken and Thompson (28) showed that the urine of rats and rabbits  injected with BAL gave a strong nitroprusside reaction within the f i r s t few hours after the injection. A similar increase i n the iodine t i t r e was also observed.  The nitroprusside test became negative and the iodine t i t r e  returned to normal within a few hours.  In order to determine whether the  excreted t h i o l was BAL, i t was precipitated as the thallous salt from the urine of rabbits injected with BAL.  The absorption spectrum of  the regenerated t h i o l was then compared with that of BAL.  It was  - 9concluded that the excreted t h i o l was very similar to BAL but not identical With-4tiX3bmparisons of the partition coefficients of the excreted t h i o l and pure BAL showed that only 50$ of the excreted t h i o l passed into the benzene layer of a benzene-water system on the f i r s t extraction, compared to 90% when pure BAL was used. However, the excreted t h i o l was able to protect the pyruvate oxidase system from inhibition by lewisite at a concentration which a l l monothiols tested were completely ineffective. It would appear, therefore, that the excreted t h i o l was a dithiol (29). If one assumes that the excreted thiol i s a dithiol similar to BAL but not identical to i t , some change i n the BAL molecule must have taken place.  Such a change could only take place at the hydroxy! group  since the two t h i o l groups appear to be intact.  It i s known that alcohols  are detoxified by conjugation with glucuronic acid or sulphuric acid. Since no increase i n ethereal sulphate was observed i t was concluded that BAL i s not conjugated with sulphuric acid (30).  A large increase i n  glucuronic acid excretion was observed i n the rat but not i n the rabbit. This would seem to indicate that conjugation with glucuronic acid might have occurred i n the rat.  BAL i s not excreted as the glucuronate (16),  although the possibility s t i l l remains that i t might be excreted, at least partially, as the glucuronide. The metabolism of BAL was also studied using radioactive BAL labelled with s 3 5 .  The Oxford group found that over 40$ of the injected  S^5 a s excreted i n the f i r s t 24 hours i n the neutral sulphur fraction, W  while only 3 - 5% of the injected S35 appeared i n the inorganic sulphate  fraction.  The  content of the ethereal sulphate was a very small  fraction of the total (31). Xoung (32).  Similar results were found by Simpson and  They reported that 63 - 71$ of the injected S35 was excreted  within 24 hours i n the neutral sulphur fraction while only 3 - 11$ was excreted i n the form of sulphate.  On further studies of the neutral  sulphur fraction they were unable to detect any radioactive BAL.  This-  does not rule out the possibility that unchanged BAL i s excreted since small amounts of BAL might easily have undergone chemical changes due to oxidation. It i s evident, therefore, that due to the wide application of BAL as a chemotherapeutic agent, a more detailed knowledge of the metabolism of this compound i s required.  The experiments to be described  were carried out i n an attempt to gain more information as to the nature of the various metabolites present i n the urine following an injection of radioactive BAL.  It was hoped that the results obtained from such a  study would form the basis for future work i n the study of BAL metabolism i n the presence of arsenic and similar toxic agents. A method was developed for the preparation of S^-ML  i n which  sodium hydrogen sulphide was reacted with 2,3-dibromopropanol to form the desired compound. The radioactive sulphur was introduced as NaS3S>H which was prepared by the reduction of barium sulphate and the trapping, on acidification of the barium sulphide, of the liberated hydrogen sulphide into a sodium ethoxide solution. Using this method BAL was prepared i n yields of 40 - 45$.  The product was identified by preparation  11 of two derivatives, paper chromatography, radiochromatography and determination of the -SH content. Analytical techniques were developed for the analysis of the various sulphur fractions i n the urine. A procedure was set up f o r the precipitation and counting of the S35 present i n the various urinary fractions.  Chromatographic techniques were also developed f o r the  separation and identification of BAL by means of various colour reactions. A technique was devised for.. radiochromatography which was used i n the separation of the various metabolites of S55 labelled BAL. Metabolic studies of S'S-BAL were then carried out using the Wistar rat as the experimental animal.  The radioactive compound was  injected intraperitoneally and the rats were placed i n a metabolism cage which permitted the separate collection of feces and urine. At various times after the injection of S35-BAL the urine was collected and analysed. It was found that 60 - 80/S of the injected S^5 period following injection. the injected S35  w  a  s  a  s  excreted i n the 48 hour  In the f i r s t 6 hours approximately 40$ of  was excreted.  However, the i n i t i a l rapid excretion of  followed by a very slow excretion.  that considerable  w  It would appear, therefore,  was retained i n the tissues.  The maximum rate of total S-*. sulphur excretion occurred during 3  the f i r s t 6 hours after injection of  -BAL. A parallel rise i n the  total sulphur excretion was also observed.  In the 6 - 1 2 hour period  after injection, however, there was a rapid decrease i n the amount of  - 12 the t o t a l sulphur excretion so that after 12 hours the t o t a l sulphur had returned to the pre-injection level. The rate of excretion of inorganic s35- iphate reached a su  maximum during the f i r s t 6 hours after injection of s35-BAL. This was followed by a steady decrease i n the rate of excretion of S35. sulphate for the remainder of the experiment.  The rate of inorganic sulphate  excretion also reached a maximum during the 0 - 6 hour period following S-*5,BAL injection.  However, there was a rapid increase i n sulphate  excretion during the latter part of the experiment. In Experiment 2, approximately 4$ of the excreted i n the form of sulphate.  was  In Experiment 3, however, 10 - 19$ of the S^S  was i n the inorganic sulphate fraction. due to a very large excretion of after the injection of S^BAL.  This increase was found to be  sulphate during the f i r s t 3 hours The amount of S35 i  phate was found to be less than 0.5$.  n  the ethereal sul-  It was concluded, therefore, that  very l i t t l e injected S35-BAL was converted into ethereal sulphate. Chromatographic studies on the excreted urine showed the presence of six possible metabolites.  Whether these compounds arose  from the metabolism of BAL i n vivo or were due to the oxidation of certain metabolites after excretion i n the urine could not be determined at present.  One of these metabolites was characterized as sulphate, while  another metabolite was found to be converted by acid hydrolysis to a third compound which was also present i n the urine.  No indication of  the presence of unchanged BAL could be found and experiments were performed to show that none of the metabolites present i n the urine could have been formed by the oxidation of excreted BAL. From studies with B-glucuronidase i t would appear that no glucuronide of BAL or i t s metabolites were present i n the postinfection urine. Also no increase i n glucuronic acid could be detected i n the postinfection urine even after acid hydrolysis. These results are i n contradiction to the results reported by Spray (30), who found a rapid increase i n the glucuronic acid excretion i n rats after BAL injection.  The reason f o r this  discrepancy i s not known. However, there was found i n the postinfection urine large quantities of glucose. From the results obtained by using color reactions to detect the presence of t h i o l s , only one metabolite could definitely be shown to be a t h i o l .  This compound gave a greenish brown spot with nickel salts.  A greenish brown colour was also reported by Spray (29) to be given by the excreted dithiol isolated from rabbits urine. Whether these compounds 1  are one and the same w i l l have to be clarified by further studies.  EXPERIMENTAL. A.  METHODS I.  Radioactive Measurements a)  Precipitation of S?5  In order to measure the amount of S^5 i n biological materials, t  i t i s necessary to oxidize the material and precipitate the sulphate as the barium or benzidine salt.  Of the many oxidation procedures the method  of Pirie (33), the method of Carius (34), and the method of oxidation with concentrated n i t r i c acid and hydrogen peroxide (35) are those most often used.  In the experiments described herein, the procedure chosen was based  on that of Carius as modified by Young, Edson and McCarter (36).  The  Carius bombs were made from Pyrex glass tubing of outer diameter 25 mm. and wall thickness of 1.5 mm.  The length of the bombs varied from 30 to  40 cm. On addition of the sample which contained from 1 - 2 mg. sulphur, and 1 ml. of fuming n i t r i c acid, the open end of the bomb was sealed by drawing out a thick wall capillary and sealing this capillary with a hot flame as described by Niederl et a l (37).  The bombs were heated i n a  Carius furnace at 300° C for 4 hours, cooled and opened. The contents were then transferred to a 50 ml. beaker and evaporated to dryness by heating with an infrared lamp. The residue was dissolved i n 5 ml. of water and the desired amount of carrier sulphate was added to bring the amount of sulphur i n the solution to about 2 mg.  If the specific activity of a  compound were being measured no carrier sulphate was added. The solution was then diluted with an equal volume of 95$ ethanol and 2 ml. of  benzidine reagent (36) were added. The solution was stored i n the refrigerator for 24 hours i n order to obtain a uniform precipitate.  The  benzidine sulphate was collected by suction f i l t r a t i o n on a weighed f i l t e r paper disc using the special suction apparatus shown i n Figure 1. The TABLE I Gravimetric determination of sulphur i n cystine by the Carius oxidationbenzidine sulphate method Sulphur added as cystine (mg.)  Sulphur Calc. %  Found %  Recovery %  1.5  26.7  26.9  100.7  1.5  26.7  27.5  103.0  1.5  26.7  27.5  103.0  1.5  26.7  27.5  103.0  1.5  26.7  27.2  101.2  Average % Recovery  102.2  weighed f i l t e r paper disc (C), 2.56 cm. i n diameter, was placed i n the brass dish (D) and moistened with water. The brass dish was then set on the ground glass rim of the funnel (E) and the suction applied. The glass cylinder (A) was put i n position and clamped i n place with small springs. The solution containing the benzidine sulphate was rapidly washed into the cylinder and the precipitate washed with 35% ethanol.  The cylinder was  then removed and any bendizine sulphate adhering to the glass was transferred to the f i l t e r paper with the aid of a small amount of 95$ ethanol.  16 -  Figure 1. A B C D E F G  -  Apparatus f o r the c o l l e c t i o n of benzidene sulphate glass c y l i n d e r glass ears f o r springs Whatman No. 1 f i l t e r paper c i r c l e s brass d i s h funnel with ground glass top rubber stopper suction f l a s k  - 17 The benzidine sulphate was washed with a small portion of ether and sucked dry.  The f i l t e r paper was removed from the brass dish and dried i n a i r to  constant weight.  Table I shows the results obtained on application of this  procedure to cystine. b) Measurement of The radioactivity i n the benzidine sulphate samples was determined by placing the samples under a thin mica end-window Geiger-Muller counter attached to a scaling unit (Nuclear Instrument and Chemical Corp. Scaling Unit Model 163). Since the radiations emitted by  are of low energy (0.167 MeV  (38)), absorption corrections were necessary for a l l the counts because of variation i n sample weight.  In order to correct for self-absorption,  samples containing different weights of benzidine sulphate but the same amount of  were prepared and counted.  The counts were then expressed  as fractions of the radioactivity i n samples containing 2 mg. sulphur and a self-absorption curve was constructed by plotting the reciprocal of these fractions against the weights of benzidine sulphate.  The reason  for choosing samples with 2 mg. of sulphur i s that such samples are suff i c i e n t l y large that they can be easily f i l t e r e d and accurately weighed. In cases where the amounts of sulphur present i n the material to be oxidized were extremely small, 2 mg. sulphur i n the form of sulphate were added before precipitation.  Table I I shows the effect of the weight of benzidine  sulphate on the counts obtained from a given amount of S"'. Figure 2 shows 3  the curve obtained for the correction of self-absorption.  - 18 -  1.30  -  1.20  -  1.10  -  A 1.00  0.90  IS  20 MG.  BENZIDINE  25  30  SULPHATE  Figure 2. Self-absorption correction curve f o r the correction factor, i s the reciprocal of the observed radioactivity expressed as a fraction of the radioactivity of the samples which contain 2 mg. S.  TABLE II Self-absorption of B-particles by benzidine sulfate samples Counts per minute  Observed Radioactivity*  A =  1 observed radioactivity  S i n sample (mg.)  Benzidine Sulfate (mg.)  1.50  13.2  2775  1.09  0.92  1.75  15.6  2588  1.02  0.98  2.00  17.8  2535  1.00  1.00  2.25  19.9  2366  0.93  1.07  2.50  22.2  2248  0.89  1.12  2.75  24.5  2175  0.86  1.16  3.00  26.5  2123  0.84  1.19  3.25  28.9  2058  0.81  1.23  *  Expressed as a fraction of the radioactivity of the samples which contain 2 mg.S.  A correction for decay was also necessary since  has a half-  l i f e of 87.1 days (38). This correction was obtained by comparing the counts per minute measured from a standard at the start of an experiment and the counts per minute measured from the same standard on the day the samples were being counted. In this manner any fluctuations i n counter performance were compensated for as well as the loss of counts due to decay. Each sample, therefore, was corrected f o r background, coincidence losses, self-absorption and decay.  - 20 II.  Synthesis of S55-BAL BAL i s usually prepared by reacting 2,3-dibromopropanol with  sodium hydrogen sulphide or ammonium hydrogen sulphide i n a closed system (39)•  For optimum yields, three to four molecules of sodium hydrogen  sulphide should be present for each molecule of the dibromide (32). a)  Preparation of S55-BAL  In the preparation of sodium hydrogen sulphide the following method was used.  The dilute sulphuric acid containing the isotopic sulphur  was precipitated as barium sulphate and reduced to barium sulphide.  The  HVjS liberated on acidification of the sulphide was swept out of the reaction flask by a stream of dry nitrogen into a solution of sodium ethoxide.  This  resulted i n the formation of sodium hydrogen sulphide. Since the only source of isotopic sulphur immediately available was the sulphate, a method was developed f o r i t s reduction.  BaS35o^ was  precipitated from a dilute solution of EgP^Q^ according to the method of Vogel (40).  The next step i n the procedure required the reduction of  BaS35o^. In order to determine the optimum conditions for this reduction, several experiments were carried out with nonisotopic BaSO^. The BaSO^ was mixed with carbon black (Darco) and heated i n a furnace at 1000°C. order to determine the extent of the reduction, the cooled mixture was decomposed i n acid solution, the liberated  swept over into a sodium  ethoxide solution and the *-SH' content determined by means of iodine titration (41). Results of several experiments are shown i n Table  IH.  In  - 21 TABLE III Reduction of BaSO^ using carbon black (Darco) Experiment No.  % yield  Period of heating at 1000°C (hours)  1  0.5  23  2  1.0  25  3  1.5  27  4  2.0  24  In an attempt to increase the yield of BaS, several experiments were carried out using sugar charcoal instead of carbon black.  In these  experiments i t was found that by increasing the "sweeping out" time to TABLE 17 Reduction of BaSO^, using sugar charcoal Experiment No.  Period of heating at 1000° G (hours)  Period of sweeping with nitrogen (mins.)  % yield  1  1  15  64  2  1.5  15  67  3  2  15  66  4  2  30  92  5  2  30  90  - 22 -  30 minutes, there was an increase i n the amount of HgS collected. Table 17 shows the results obtained. Based on the above experiments, the following procedure was evolved f o r the preparation of NaS^Sn.  BaS'5o^ (0.3 gms.) was mixed intimately  with sugar charcoal (0.6 gms.), wrapped i n a f i l t e r paper, and placed i n a combustion boat. furnace.  This was then inserted i n the combustion tube of the  The furnace was heated to 1000°C and maintained at this tem-  perature for two hours.  After cooling, the BaS35 was removed from the  furnace and transferred to the reaction flask of the gas transfer apparatus.  The Bas'-* was decomposed with the calculated amount of 1 N HCI  and the HgS'^ liberated was removed from the flask by bubbling a stream of dry nitrogen through the acid solution for 30 minutes.  The H2S35 was  trapped i n a ethanol solution containing 200 mg. of sodium ethoxide. This solution was immersed i n a dry ice-alcohol bath i n order to obtain maximum absorption of HgS^S. The solution was then set aside i n an i c e bath u n t i l required for the next stage of the synthesis. The BAL was prepared by a modification of the method of Simpson and Young (32).  Freshly d i s t i l l e d methanol (40 ml.) was placed i n a  500 ml. pressure flask and 3 gm. of freshly cut sodium were added.  The  flask was placed i n an ice-bath and dry HgS was passed i n under pressure for six hours.  To this solution were added 3 gm. of 2:3-dibromopropanol  (Eastman Kodak Co.) and then the cold solution of isotopic NaS35H. flask was sealed with a rubber stopper which was wired i n place,and  The  - 23 the solution heated i n a water-bath at 60° C for 80 hours.  After this  time, a white precipitate of sodium bromide covered the bottom of the flask.  The flask was cooled and opened; one drop of Congo red indicator  solution was added and concentrated hydrochloric acid was added slowly until a blue end-point was reached. containing isotopic  The excess hydrogen sulphide,  was swept by a stream of nitrogen into a centri-  fuge tube containing sodium ethoxide.  The methanol was removed by  evaporation under reduced pressure at 40° C.  The moist residue remaining  was dissolved i n 40 ml. of d i s t i l l e d water and an o i l separated out.  The  solution was extracted with five 30 ml. portions of freshly d i s t i l l e d , peroxide-free ether, and the ether extract was dried over anhydrous sodium sulphate.  The ether solution was evaporated under reduced pressure to a  small volume. This was transferred, with the aid of a small amount of dry, peroxide-free ether into a 10 ml. d i s t i l l i n g flask, which was set up for vacuum d i s t i l l a t i o n .  The system was slowly evacuated until the pres-  sure was such that the ether i n the flask began to d i s t i l .  On removal of  the ether the system was evacuated to a pressure of approximately .025 of Hg and the contents of the flask were slowly heated.  mm.  Any d i s t i l l a t e  collected below 50° C was discarded. At 62° - 64° C the BAL d i s t i l l e d over and a 40$ yield was obtained, based on the weight of 2:3-dibromopropanol used. The '-SH' content was normally between 52 - 53$ as determined by the method of Simpson, Zbarsky and Xoung (42) i n which an acid solution of BAL was titrated with standard KIOj i n the presence of excess KI.  The calculated '-SH' i s 53.2$. However, i f the »-SH* content  - 24 f e l l below 52% the BAL was r e d i s t i l l e d to give a yield of 27 - 50%. b)  Characterization of S35-BAL  In addition to the determination of the sulfhydryl content, the following methods were used to characterize the BAL. (i)  2:2-dimethyl-4-hydroxymethyl-l:3-dithiolan  CH SH 2  / C  CHSH  +  H  2  —  0 = C  I  CH -S  3  2  CH  > CH - S ^  ^  3  + HgO  I  CHJ  CH 0H  X c /  CH OH 2  BAL (0.62.gm.) and acetone (4 ml.) were dissolved i n 8 ml. of benzene. To this solution were added 3 drops of concentrated hydrochloric acid.  After heating under reflux, for 10 hours, at 65° C, the solution  was dried over anhydrous sodium sulphate. The solution was then evaporated to a viscous o i l containing the dithiolan.  The dithiolan was crystallized  from a benzene-petroleum ether (boiling range 30° - 60° C) solution. The product obtained (m.p. 49° - 51° C) was recrystallized to give 0.6 gm. of pure white crystals melting at 55° - 55.5° C. The melting point reported by Stocken (15) for this compound i s 54° - 55° C. (ii)  2-phenyI-4-hydroxymethyl-l?3-dithiolan  CH SH  I  2  CHSH CH 0H 2  +  0 II HC—  CH« -  I  > CH - S ^  C H  X  + HgO  CH 0H 2  BAL (0.33 gm.) and benzaldehyde (1 ml.) were dissolved i n 20 ml.  ~ 25 »  of benzene. To this solution were added 3 drops of concentrated hydrochloric acid.  The solution was heated for 6 hours at 65° C during which  time the solution turned clear and an aqueous layer formed on the bottom of the flask.  The benzene layer was removed, evaporated u n t i l a clear  viscous liquid remained and the dithiolan crystallized from a benzenepetroleum ether (boiling range 30° - 60° C) solution.  The product formed  was recrystallized. to give 0.45 gm. of white crystals with a melting point of 67° - 68° C, as compared with a melting point of 66° - 68° C reported by Simpson and Xoung (32).  The sulphur content was found to be 30.0$,  30.2$ (Theoretical 30.2$) (iii)  Chromatography  The synthetic BAL was compared with an authentic sample of BAL by means of f i l t e r paper chromatography. A pure sample of BAL gave only one radioactive spot when run i n a tert.-butanol-water solvent (70/35; V/V).  The. BAL was also detected by i t s a b i l i t y to form a chocolate brown  complex when treated with a 1$ nickel chloride solution.  A detailed  description of the chromatographic techniques i s given i n Section IV". In a typical experiment the following results were obtained from characterization studies of the prepared BAL. found to be 52.8$, 52.9$ (theor. 53.2$).  The  The sulfhydryl content was 2:2-dimethyl-4-hydroxymethyl-  li3-dithiolan had a melting point of 55° - 55.5° C (literature 54° - 55°C). The 2-phenyl-4-hydroxymethyl-l:3-dithiolan had a melting point of 67° - 68° C.  (literature 66° - 68° C) and a sulphur content of 30.0$, 30.2$  -  (theoretical 3 0 . 2 $ ) .  26  A portion of the  -  2-phenyl-4-hydroxymethyl~l:3-  dithiolan was analysed for i t s §35 content and was found to have a specific activity of 3012 counts/mg.S. An aliquot of the original BAL on analysis for S^5 was found to have a specific activity of 3000 counts/mg.S,.' Chromatographic studies showed only one radioactive component was present. This component had an %  value identical with that of the authentic sample  of radioactive BAL run on the same strip of f i l t e r paper. When the chromatogram was dipped i n a 1$ nickel chloride solution a chocolate brown spot was obtained with an Rf value identical with that obtained from authentic BAL. III.  Urinary Sulphur Analysis In most urinary studies concerned with sulphur metabolism the  sulphur present i n the urine i s classified into two groups; the unoxidized or neutral sulphur and the oxidized sulphur or sulphate ( 4 3 ) .  The latter  can be further subdivided into inorganic sulphate and ethereal or conjugated sulphate. Ethereal sulphate can be converted into inorganic sulphate by acid hydrolysis. a)  Total Sulphur  The total sulphur was determined by oxidizing the urine sample with fuming nitric acid and precipitating the benzidine sulphate as described previously. b)  Total Sulphate  In Experiment 2 the total sulphate was determined using the method described by Simpson and Xoung (32) i n which the urine was evaporated to  dryness i n the presence of hydrochloric acid and the S^-> content of the supernatent f l u i d and of the residue was determined.  This method, however,  was found to be unsatisfactory since the nature of the sulphur present i n the insoluble residue could not be determined.  An additional disadvantage  was that the benzidine sulphate precipitates formed by this method gave very uneven surfaces. Therefore, i t was very d i f f i c u l t to obtain reproducable counts on duplicate samples even though the weights were identical i n each case. A new method for total sulphate was developed to overcome the factors mentioned above. This method was based on the work of Owen (44), and McKittrick and Schmidt (45). For accurate determinations of sulphate as the benzidine salt i t i s necessary to remove any phosphate that may be present (46). However, even i f as much as 2 mg. of phosphorous were present for each mg. of sulphur, the increase i n weight of the benzidine precipitate would only be equivalent to 0.07 mg. of sulphur (36).  Since  the amount of sulphur present i n the samples to be analysed was usually less than 1 mg., any interference by phosphorous would have very l i t t l e influence on the weight of the benzidine precipitate. The procedure used to determine the t o t a l sulphate i n Experiment 3 was as follows. To 1 ml. of the urine solution was added 2 ml. of carrier sulphate and 0.5 ml. of 3 N hydrochloric acid.  This solution was heated  i n a sand-bath almost to dryness, cooled and 5 ml. of water were added to dissolve any residue. Dilute sodium hydroxide solution was added, drop by  - 28 drop to the bromo-phenol blue end-point.  Dilute hydrochloric acid was  then added to bring the solution back to the acid side of the bromophenol blue end-point.  At this pH, approximately 2.8, benzidine sulphate  has i t s minimum solubility (45). An equal volume of 95$ ethanol and 2 ml. of benzidine reagent (36) were added and the solution was stored  i n the  refrigerator for 24 hours i n order to obtain a suitable precipitate.  The  solution was then filtered and the precipitate was collected on a weighed TABLE Y Recovery of added S35Q^ i n the determination of total sulphate Sample No.  Wt. benzidine sulphate (mg.)  S 0 ~ added (c.p.m.) 3 5  4  S^o^"" recovered (c.p.m.)  % recovery  T  l  17.1  6303  6240  99.0  T  2  17.4  6303  6301  100.0  h  13.6  5931 .  5943  100.2  4  13.5  5931  5929  100.0  T  f i l t e r paper i n the usual manner. On drying, the precipitate was weighed and the radioactivity determined. In order to check the above procedure, the following experiment was carried out.  A known amount of isotopic sulphur was added as inorganic  sulphate to non-radioactive urine. The total sulphate was then determined i n the usual manner and the precipitate was weighed and the activity  - 29 determined. c)  The results obtained are given i n Table V. Inorganic Sulphate  In Experiment 3 the inorganic sulphate was determined i n a manner similar to that described above for total sulphate except that no acid hydrolysis was performed. The urine sample was adjusted to pH 2.8 using TABLE VI Recovery of added . s'5()/~ i n the determination of inorganic sulphate Solution No. Wt. Benzidine sulphate (mg.)  S 0 ~ added (c.p.m.) 3 5  4  S ^ o ^ recovered (c.p.m.) 3  % recovery  Il  27.1  6396  6416  100.3  *2  27,0  6396  6577  102.8  h h  27.0  6396  6384  99.8  26.9  6396  6577  102.8  16.6  6303  6241  99.0  16.6  6303  6322  100.3  X  5  bromo-phenol blue as indicator.  To this solution was added 2 ml. of  carrier sulphate, 3 ml. of 35% ethanol and 2 ml. of benzidine reagent. The solution was stored i n the refrigerator for 24 hours after which time i t was f i l t e r e d on a weighed f i l t e r paper disc using the apparatus described previously.  The precipitate was dried, weighed and the radio-  activity determined i n the usual manner. The results obtained using this  procedure on non-radioactive urine to which, a known amount of S ^ S Q ^  was  added, are given i n Table 7 1 . 37.  Chromatography Since paper partition chromatography was f i r s t introduced by Martin  and Synge (47) i t has found wide use i n the separation of urinary constituents.  As a major portion of the experimental work to be discussed was  concerned with the number and nature of the urinary metabolites arising from the injected radioactive BAL, a method was developed f o r the detection and separation of these metabolites. a)  Detection of BAL and i t s metabolites by means of color reactions  A suitable method was developed for the detection of micro amounts of BAL and similar thiols on a f i l t e r paper chromatogram. The chromatograms were run on Whatman No. 1 f i l t e r paper and best results were obtained by using a tert.^butanol-water solvent and locating the BAL on the paper by moistening with a 1% solution of nickel chloride i n water. The following chromatographic method was found to give the most uniform results.  The chromatograms were run on strips of Whatman No. 1  f i l t e r paper, 15 cm. x 57 cm. means of a micropipette.  The sample was applied as a small spot by  If i t were necessary to apply a relatively large  amount of solution, several drops were added on the same spot, the spot being dried with a stream of nitrogen after each application.  The s t r i p  was then placed immediately i n the chromatography jar and arranged f o r descending chromatography using a tert.-butanol-water solvent. (70/35;7/7)  - 31 and the jar made a i r tight.  After 24 hours the paper strip was removed.  If the chromatogram were being tested for the presence of sulphydryl compounds the paper was dipped ^rhile s t i l l wet, i n the reagent, since i t was shown that BAL and other thiols were oxidized quite rapidly i f the paper were l e f t i n the a i r to dry. Choice of a nickel solution as a reagent for the location of BAL on f i l t e r paper chromatograms was based on the findings of Spray, Stocken and Thompson (29) that BAL reacted with metalisalts to give colour complexes.  It was found that by dipping the chromatogram into a 1% solution  of nickel (ous) chloride, as l i t t l e as 8 ug. of BAL could be detected. The presence of BAL was indicated by the formation of a chocolate brown spot on the paper chromatogram. Mono-thiols, such as cysteine, could only be detected i n much larger amounts. The presence of cysteine was indicated by the formation of a green spot, after the paper had dried for a few hours. The possible use of the nitroprusside- reagent of Toennies and Kolb (48) was also investigated as a means for detecting BAL on a f i l t e r paper chromatogram. On dipping the chromatogram i n this reagent, a b r i l l i a n t red colour appeared at once on the BAL spots but faded within a few minutes.  On drying, however, a permanent light blue spot appeared.  After a few days, the entire paper slowly turned a bluish green but the blue spots were s t i l l detectable*. The b r i l l i a n t red spot and subsequent  *  These results differ from those obtained by Toennies and Kolb who found  - 32 -  blue spot were obtained for both BAL and cysteine with concentrations as low as 8 jag. The following reagents were also used i n the chromatographic studies of the urinary metabolites of BAL. Benzidine reagent (49) was used for the detection of inorganic salts and certain reducing compounds, bromo-cresol green reagent (50) f o r the detection of acids and bases, and ammoniacal silver nitrate reagent (50) for the detection of sugars. Several solvents were tested before deciding on the use of the tert.-butanol-water solvent (70/35).  These solvent mixtures and the  results obtained are given i n Table VII.  Each solvent was tested i n  the following manner. A small strip of f i l t e r paper containing a spot of BAL was placed i n a large test tube containing a few ml. of the solvent.  The test tube was sealed with a cork and ascending chromato-  graphy was carried out.  After a few hours the paper strip was removed  and treated with 1% nickel solution i n order to detect the BAL spot. More detailed studies were carried out on Solvents No. 5, 6 and 8. Solvent No. 5, on a large strip of f i l t e r paper, gave a spread out spot of BAL.  In many cases no spot at a l l could be detected.  The reason  for this i s not known. Solvent No. 8 was f i n a l l y chosen since the BAL  that the red color "remained intact for weeks" (48) against the green background. In one preparation of this.reagent the red color did remain intact but i n a l l other preparations the blue color was obtained. This discrepancy was perhaps due to a slight difference i n the amounts present of the various components used to prepare this reagent.  - 33 spots were very well defined with no indication of spreading.  TABLE VII Ascending chromatography of BAL using various solvent mixtures Solvent  Nature of spot obtained with 1% N i C l  (VA)  2  Poorly defined, faint color  .95  Poorly defined, faint color  .90  25 75  Poorly defined, faint color  .85  70 30  No spot obtained  1. Ethanol H0  75 25  2. Ethanol H0  50  3. Ethanol H0  2  2  2  4.  Phenol H0 c.NH^OH 2  Approximate %  50  '  -  4  Fairly well defined, distinct color  .95  6. n-butanol saturated with B^O  Well defined, distinct color  .90  7. Tert.-butanol 50 H0 30  Well defined, distinct color  .90  8. Tert,-butanol 70 H0 35  Very well defined, distinct color  .90  5. Phenol  80 20  2  2  In experiments where the presence of sugars was being studied, the solvent system used was n-butanol-glacial acetic acid-water (40Ao/50)(5l).  - 34 b)  Detection of BAL and i t s metabolites by means of radioactivity  The use of paper partition chromatography for the detection and separation of radioactive urinary constituents has proved to be quite successful for the study of radioactive sulphur compounds (52, 53).  The great  advantage i n the use of radioactivity to detect the presence of urinary metabolites is that any metabolite which arises from the injected radioactive compound can be detected, irrespective of i t s chemical nature, by the presence of the radioactivity.  Therefore, a method was developed for  the detection of isotopic sulphur on a f i l t e r paper chromatogram. For the preparation of the radiochromatograms the method used was similar to that given above in, a).  On removal of the f i l t e r paper strip  from the chromatography jar, the paper was dried. A strip, 1 1/8" wide, containing the path followed by the urine spot, was cut from the larger piece of the f i l t e r paper and this strip was cut into 1 cm. sections, starting from the point of application of the original spot and  extending  to the solvent front. Each strip was then counted under a Geiger-Muller tube.  The counts per minute were plotted against the distance the p a r t i -  cular section was from the point of application of the original spot.  B. METABOLISM EXPERIMENTS I.  Experiment 1  Before studying the urinary excretion products of BAL metabolism, i t was desirable to determine to what extent BAL would undergo oxidation i n urine at room temperature. Simpson and Xoung (32) had reported that BAL was oxidized quite rapidly i n urine at a neutral pH but to a lesser extent i n acidified urine.  Urine, therefore, was collected from three TABLE VIII  Rate of oxidation of BAL i n acidified and neutral urine Time  % of i n i t i a l BAL remaining i n Acidified urine  Time  Neutral urine  % of i n i t i a l BAL remaining i n Acidified urine  100  100  10 hours  94  18 mins.  99  97  17 hours  82.5  35 mins.  98.5  92  25 hours  81.0  60 mins.  98  86  70 hours  62.1  4 hours  95  61  94 hours  55.5  8 hours  94.5  58  0 min.  Neutral urine 38  -  male white rats of the Wistar strain under the. same conditions that prevailed i n the remaining experiments. The urine was f i l t e r e d and divided into two parts, one half being acidified to pH 2 with hydrochloric acid,  -36-  1  1  1  •—Q  1  o o  ,75  _______  o  —  < m <  1  1  • \ 50  •  Z u. O  -  —  25  »* 0  1 0  i  1  1  1  1  •  5  10  15  20  25  30  TIME  IN  HOURS  Figure 3. Rate of oxidation of BAL i n neutral urine and i n acidified urine as determined by iodine titration. • - BAL i n neutral urine. o — BAL i n acidified urine.  while the ether portion was l e f t at the original pH.  BAL was then added  u n t i l each solution contained one mg. BAL/ml. urine and at various time intervals, aliquots of each sample of urine were tested for the amount of BAL present using the iodine t i t r a t i o n method of Simpson, Zbarsky and Xoung (42).  The results obtained are given i n Table VIII and Figure 3.  It was found that the oxidation of BAL could be reduced to a minimum bycollecting the excreted urine i n a flask containing sufficient 6 N hydrochloric acid to prevent the pH of the urine rising above pH 2. U.  Experiment 2  Although the rate of excretion of S 5 i n the urine of rats i n 3  jected with radioactive S^-BAL has been studied by various workers (31, 32), no detailed study as to the number and nature of the various metabolites has been reported.  The following experiment, was therefore  carried out i n order to determine the nature of the various metabolites, using the chromatographic techniques described above. A more detailed study of the rate of excretion of S ^ i n the various sulphur fractions was 3  also carried out. In this experiment, six male, white rats of the Wistar strain were injected with S 5_BAL. The rats were divided into two groups of three, 3  group A having a combined weight of 737 gm., and group B, 796 gm.  Each  rat received by intraperitoneal injection, 20.3 mg. of BAL dissolved i n 0,5 ml. of propylene glycol. 9,100  Since the BAL had a specific activity of  c.p.m./mg. BAL, each rat received 184,700 c.p.m., or each group  38 received approximately 554,100 c.p.m. Each group of rats was placed i n a metabolism cage which permitted the collection of feces separate from urine.  The animals were allowed water ad libitum but were given no food  while i n the cage; instead they'were fed i n a different cage for approximately two hour intervals every 12 hours.  The urine excreted during the  feeding period was collected on wax paper placed under the cage and was added to the main sample f o r that particular time period. regular collection periods, 0 - 3 ,  3-  During the  6, 6 - 12, 12 - 24, 24 - 48 hours,  the urine was collected i n dilute acid. Each collection was f i l t e r e d and stored i n the deep freezer. At the end of 48 hours, the animals were sacrificed and the carcasses stored i n the deep freezer f o r future study. In the f i r s t hour after injection a l l animals showed signs of lachrimation while two of the rats i n group A had mild convulsive seizures. However, since these animals received an amount of BAL equivalent to 2/3 L.D. 50 (16) i t i s not suprising that the convulsive seizures were observed. Since such a large dose,of BAL was required, the animals were placed i n a cool room immediately after injection as i t had been reported by McDonald (54) that the mortality rate of rats injected with BAL was at a minimal at 17.2° C.  After the f i r s t few hours, a l l the animals returned to normal,  although the animals i n group B were very i r r i t a b l e . a)  Nitroprusside test  It had been reported by Stocken and Thompson (28) that a strong positive nitroprusside reaction was observed i n the urine for the f i r s t six hours following the injection of BAL i n the rabbit.  In order to  -  39 -  determine whether a similar result occurred i n the rat, the following experiment was carried out. 3-6  From both group A and B, 0.5 ml. of the 0 - 3 ,  and 6 - 1 2 hour urine samples were diluted to 1.5 ml. and 2 drops of  nitroprusside reagent (48) were added. The color obtained was compared with a blank containing d i s t i l l e d water and nitroprusside, and a standard solution of BAL prepared by diluting 1 drop of a 1 mg. BAL/ml. solution to 1.5 ml. with d i s t i l l e d water and 2 drops of the nitroprusside reagent.  In the  0 - 3 , 3 - 6 and 6 - 1 2 hour urine samples, a positive nitroprusside test was obtained indicating that the urinary excretion of thiols increased rapidly after BAL injection. hour period.  The maximum colour was reached i n the 3 - 6  Both the preinjection urine and the urine samples collected  12 hours after the injection showed negative results. b)  Chromatographic studies  Chromatograms were run on a l l the urine samples collected, using the method described previously. The solvent used was tert.-butanol-water (70/40). strip.  Twenty jal of urine were applied to each spot on the f i l t e r paper On removal of the papers from the chromatography jar the chromato-  grams were tested with the various reagents discussed above.  Radioactive  measurements were also made using the techniques described previously. The results obtained are described below. i)  Color reactions Hickel reagent  -  On dipping the chromatograms i n the 1%  nickel chloride solution no spots were obtained except the BAL standard  - 40 which gave a chocolate brown spot with an  value of 0.92.  On drying,  however, a faint greenish-brown spot appeared i n the 3 - 6, hour urine sample of group A.  This compound had an Rf value of 0.70.. Cysteine, a  monothiol, when run on the same paper, as a standard, also produced a light green spot when dry.  This spot had an Rf value of 0.20.  However, no spots  were obtained i n any of the urine samples which corresponded to the Rf value of BAL or cysteine. Nitroprusside reagent  -  When the chromatograms were dipped i n  nitroprusside, no spots were obtained except for the BAL standard (Rf — 0.91). However, on drying, several blue spots were obtained.  A blue  spot (Rf = 0.47 - 0.51) was present i n a l l the urine samples.  The presence  of several other blue spots was indicated but no Rf value could be obtained as no well defined spot was observed but rather a defused band of colour with areas of varying intensity.  In the 3 - 6  hour urine sample from group  A, a well defined blue spot was obtained with an Rf value of 0.69. Benzidine reagent  -  It had been reported by Spray (30) that there  was a marked rise i n the urinary glucuronic acid output i n the rat after injection of BAL.  In order to determine whether a similar excretion had  occurred during the experiment described herein, a chromatogram containing the urine samples was sprayed with benzidine Beagent (36) which w i l l detect the presence of reducing substances (49). A large brown spot having an Rf value identical to that of the glucose standard was found i n the 0 - 3 the 3 - 6  hour urine samples of both groups.  In the 6-12  and 12 - 24  and  hour samples this compound could s t i l l be detected although the amount present was greatly diminished as compared with the 0 - 3 and 3 - 6 hour samples. ii)  Radioehromatograms  The results obtained from the radioehromatograms are shown i n Figure 4 and 5. The amount of radioactivity as counts per minute was plotted against Rf value.  The presence of five urinary metabolites of  BAL and the possibility of a sixth was indicated by the results obtained. These compounds showed the following Rf valuesj* Compound 1  Rf  0.00 - 0.05  Compound 2  R  f  0.20 - 0.27  Compound 3  Rf  0.35 - 0.41  Compound 4  Rf  0.54 - 0.60  Compound 5  Rf  0.68 - 0.74  Compound 6  Rf  0.92 - 0.98  In group A the greatest excretion of compound 1 occurred during the f i r s t three hours after the injection and slowly decreased until after 24 hours the amount of this compound present i n the urine was just large enough to be detected.  Compound 3 was present i n a l l urine samples except  the 3 - 6 hour sample.  In the 3 - 6 hour sample there was a very large  increase i n compound 5, suggesting that compound 3 may have been converted to compound 5 during the collection of the urine. Compound 4 was present  * Since the chromatograms were cut into cm. strips the Rf values i s given as an R^. range.  4  5  Figure 4. Radiochromatograms of urine from group A r a t s injected i n t r a p e r i t o n e a l l y with S BAL. Solvent t e r t . butanol-water (70/40). The numerals above the peaks indicate the number of the compound. Bracketed numerals indicate doubtful peaks. 53  I  1  (27)  (26)  123)  •  Cb  O  0  0.5  IX)  <  0  3 (24)  O  4  4  (40)  <23i  •  P  0.5  IJO  0  0.5  10  Figure 5. Radiochromatograms of urine from group B rats injected intraperitoneally with s35-BAL. Solvent tert. butanol-water (70/40). The numerals above the peaks indicate the.number of the compound. Bracketed numerals indicate doubtful peaks.  i n a l l the urine samples i n quite large amounts and appeared to be the major metabolite.  Compound 5 was present i n small amounts i n a l l the  urine samples except during the 3 - 6 hour period, when, as was mentioned above, a large amount of this compound was present.  It was also found that  compound 5 was present ingreater amounts whenever the concentration of compound 3 bad decreased to a small amount as was found i n the 12 - 24 and the 24 - 48 hour samples.  Compound 6 did not appear i n the urine u n t i l  six hours after the injection and was found i n small amounts for the remainder of the experiment. The possible presence of another metabolite, compound 2, was indicated i n the 0 - 3 hour sample and i n the 12 - 24 and 24 - 48 hour samples. However, during the remainder of the experiment no indication of this compound was found. The BAL standard was found to have an Rf> range of 0.87 - 0.93. In group B similar results were obtained with the following exception.  In the 0 - 3 hour period a single compound was found having an  Rf range between compounds 3 and 4. found i n this urine sample.  No indication of compound 3 or 4 was  Therefore, i t was d i f f i c u l t to determine  whether this compound was due to the fusion of ^ :peaksnew metabolite.  :  3 and 4, or to a  In the 3 - 6 hour sample, however, this compound had  disappeared and compounds 3 and 4 were present. A summary of the radioactive metabolites found i n the urine of groups A and B i s given i n Table EC. *  . - 45 TABLE IX Radioactive metabolites of BAL present i n the urine of groups A and B Urine sample Compound  Rf range  Time period Group  0-3 hr. 3-6 hr. 6-12 hr, 12-24 hr. 24-48 hr. A  B  A  B  A  B  A  B  A  B  1  0.00-0,05  + +  + +  +  +  +  +  2  0.20-0.27  + -  .- -  -  -  +  +?  +  3  0.35-0.41  + *  - +  +  +  +  +?  +? +  "4  0.54-0.60  + +  +  +  +  +  +  +  5  0.68-0.74  + +  + +  +? +  +  -  +  -  6  0.92-0.98  - -  - +  +  +  +?  +  +  +  +? +? +  + - Compound present i n urine sample - - Compound not present i n urine sample +? - Presence of compound indicated, but the peak not large enough to be classified as + * - One peak with Rf range 0.45 - 0.55 The possibility that some of the metabolites present i n the postinjection urine might have arisen from the oxidation of excreted BAL gave rise to the following experiment. Urine was collected from 3 male Wistar rats and adjusted to the same pH as the urine samples collected from the rats injected with the radioactive BAL. A known amount of S^^.BAL was added to this urine so that the amount of S^/ml, urine was similar to the amount found i n the urine collected during the 3 - 6 hour period i n the experiment where radioactive BAL was injected. At 0, 3, 6, 9, 12,24 and  Figure 6. Radioehromatograms of rat urine containing added -'-BAL. 40^1. of urine were run on paper chromatograms at 0, 3, 6, 9, 12, 24 and 48 hours after the S35-RAL was added to the urine. The radiochromatogram of the 0 hour sample i s not shown i n the figure but i s identical to the others.  - 47 48 hours 40 l.of the urine were chromatographed. The chromatograms were then cut into 1 cm. strips and the radioactivity determined.  The results  obtained are shown i n Figure 6. Chromatograms were also treated with 1% nickel chloride and with nitroprusside i n order to determine whether the TABLE X The detection of BAL on a f i l t e r paper chromatogram with 1% nickel chloride and with nitroprusside. BAL was added to rat urine and chromatograms of the urine were run at various time intervals Time (hours)  Amount of urine applied to f i l t e r paper strip (/*1.)  The presence of BAL as indicated by Nickel reagent  Nitroprusside reagent  0  20  3  40  +  +  7  40  +  +  12  40  +  +  24  40  mm  48  40  met  -  amount of BAL present i n the urine at the various times could be detected on the f i l t e r paper strip.  The results obtained are shown i n Table X.  The results shown i n Figure 6 indicate that even after 48 hours no breakdown products of BAL could be detected i n the urine.  This would  seem to indicate that none of the radioactive components present i n the  -  48  postinfection urine arose from the breakdown of any BAL that might have been present i n the urine.  Although the size of the radioactive peak  obtained on the radioehromatograms remained f a i r l y constant throughout the 48 hour period, the nickel reagent and the nitroprusside reagent were unable to detect the presence of BAL i n the urine after 12 hours. c)  Sulphur analysis (i)  Total sulphur The total sulphur and the total s35„ iphur were determined 3u  for each urine sample using the method described previously. obtained are shown i n Table XI.  The results  Since each urine sample was diluted to  some extent.by drops from the drinking water, the amount of S^/mi. urine did not give a true indication of the rate of BAL metabolism. For this reason the amounts of sulphur/hour and the amounts of S^^/hour were calculated.  The results obtained are shown i n Figure 7.  The rate of ex-  cretion of the injected S'5 i n the urine is shown i n Figure 8. (ii)  Total sulphate The total sulphate content of each urine sample was  determined using the method of Simpson and Young (32). The results obtained are given i n Table XII.  '  The insoluble residue obtained on the  acid hydrolysis was oxidized and the sulphur and S-^-sulphur content determined. The results obtained are given i n Table XIII. The rates of excretion of the total sulphate and t o t a l s35sulphate at the various time intervals during the experiment are given i n Figure 9.  TABLE XI Total sulphur and total s35_ iphur i n rat urine after intraperitoneal injection of 554,100 c.p.m. of S"*BAL/group of three rats 3  3u  Group  A  B  Time period after i n ject ion (hrs. )  Total S Volume of urine ex(mg.)/ creted (ml. ) ml.urine  Total S Total S excreted (mg.)/ during time nr. period(mg.)  Total S 5 Total S Total S (c.p.m.)/ (c.p.m.)/ excreted ml. urine during time hr. period(cpm) 35  3  55  0-3  11.5  1.13  13.0  4.3  11778  135,447  45,147  3-6  6.5  2.52  16.4  5.7  13068  110,279  36,760  6-12  7.6  2.03  15.4  2.6  7491  56,930  9,490  12-24  57.5  0.39  22.4  1.9  996  57,270  4,772  24 - 48  28.0  1.00  28.0  1.2  21,812 381,740  909  0-3  15.5  1.02  15.8  5.3  8,873  137,531  45,844  3-6  9.5  2.23  21.2  17,026  161,747  53,916  6-12  18.5  0.85  15.7  2.6  3,854  71,299  11,883  12 - 24  65.0  0.42  27.3  2.3  1,013  65,845  5,487  24 - 48  40,0  0.84  33.6  1.4  236  9,440  393  Total  - 445,862  779  Total  -  - 50 -  K  6 0 P O 0  GP. A  GP.  A  X •j  6  30,000  et I  40,000  K  Q. _l  XJ a.  (0 30,000 I  3  3  _i  3  in  S  J 20,000  <  o  o  10,OOO  Q  0 - 3 3 - 6 6-12 12-2424-48 TIME PERIOD HRS. (7.1)  0 - 3 3 - 6 6-1212-2424-48 TIME  PERIOD H R S .  ce 60,000 GP. B  ct  x.  V  GP. B  s  a: 50,000  6  §40,000  at  3 X  »3O,0O0  3 U>  CO  _j 20,000 <  o  o K  H  I  10,000  'mm 0 - 3 3 - 6 6-12 12-2424-48 TIME PERIOD HRS.  0 - 3 3—6 6-12 12-242*48 TIME  PERIOD HRS.  Figure 7. Rate of excretion of total sulfur and total s25-sulphur at various time intervals following intraperitoneal injection of S -BAL into the r a t . 55  51  Figure 8. Excretion of i n the urine of rats injected intraperitoneally with S35_BAL. O - group B • - group A  TABLE XII Total sulphate and total s'^-sulphate i n rat urine after intraperitoneal injection of 554,100 c.p.m. of S BAL/group of three rats Group  A  B  Volume of Time period urine exafter i n ject ion(hrs.) creted (ml. )  Total S O ^ Total SO4 Total S 0 (mg.)/ml. excreted (mg.)/ during time hrs. urine period(mg.) 4  Total s55o. "(c.p.m.)/ ml. urine  Total S 5 Q excreted during time period(cpm) 5  4  11.5  0.13  1.49  0.50  288  3310  1100  3 - 6  6.5  0.33  2.14  0.71  688  4460  1490  6-12  7.6  0.38  2.89  0.48  404  3075  510  12-24  57.5  0.13  7.48  0.62  61  3500  290  24 - 48  28.0  0.53  2600  110  0.62  93 Total  -  16,945  0 - 3  15.5  0.09  1.39  0.46  221  3420  1140  3 - 6  9.5  0.25  2.38  0.79  467  3440  1480  6-12  18.5  0.23  4.25  0.71  243  4500  750  12 - 24  65.0  0.16  10.4  0.87  77  5010  420  24 - 48  40.0  0.38  15.2  0.63  31  1240  50  Total  - 17,610  3  -  Total s35o (c.p.m.)/ nr.  0 - 3  14.9  3  4  TABLE XIII Residual sulphur and residual S -*_ ulphur i n rat urine after acid hydrolysis. Each group of three rats received 554,100 c.p.m. of s35_BAL intraperitoneally 3  S  Group Time period after i n jection(hrs«)  Residual Rgsidual S35 exS (cpm)/ creted hr. during time period(cpm) 55  0.65  2825  32,500  10,800  0.22  579  3,760  1,250  0.30  0.05  25  190  30  1.73  0.14  #  #  #•  *  2.94  0.98  2277  35,250  10,750  0.16  1.52  0.51  1451  13,800  4,600  18.5  0.06  1.11  0.18  28  518  86  12 - 24  65.0  0.07  4.55  0.38  24 - 48  40.0  0.07  2.80  0.12  0.17  1.95  6  6.5  0.10  0.65  6 -.12  7.6  0,04  12 - 24  57.5  0.03  24-48  28.0  0-3  13.5  0.19  3-6  9.5  6-12  3 -  B  cpm  11.5  0-5  A  Residual S Residual Residual S Residual S Volume of (mg.)/ml. excreted (mg.)/hr. S35( )/ urine exduring time creted (ml. ) urine ml. urine period (mg.)  # *  #  weight too small to measure activity too small to measure  #  •  * #  #  •*  TIME  PERIOD  HRS.  TIME  PERIOD  HRS.  Figure 9. Rate of excretion of t o t a l sulphate and t o t a l S35-sulphate at various time intervals following intraperitoneal injection of S35-BAL into the r a t .  - 55 III.  Experiment 5  In Experiment 3 an attempt was made to check the results obtained i n Experiment 2. A more detailed stucty- as to the nature of the various metabolites was also undertaken. In this experiment six female, white, Wistar rats were injected 35 with S^ -BAL. As i n the last experiment, the rats were divided into groups of three, group D having a combined weight of 585 gm. while group C weighed 591 gm. Each group received by intraperitoneal injection, 49.4 mg. of BAL dissolved i n propylene glycol (68.6 mg. BAL/ml. propylene glycol). Since the BAL had a specific activity of 5,000 c.p.m./mg. BAL, each group received 247,000 c.p.m. The rats were placed i n a metabolism cage and the urine was collected i n the manner described i n Experiment 2. At the end of 48 hours the animals were sacrificed and the carcasses stored i n the deep freeze f o r future study. As i n Experiment 2, a l l the animals saowed signs of lachrimation after the injection of the BAL. By the end of the f i r s t hour a l l animals had experienced quite severe convulsive seizures.  However, these seizures  soon passed and after 3 hours the animals appeared to be normal. For the remainder of the experiment, the rats were quite active and showed no signs of fighting unlike the male rats used i n Experiment 2. a) Chromatographic studies Chromatograms of a l l the urine samples were run using the same method as described i n Experiment 2. Chromatograms of pre-injection urine were also run as controls.  The solvents used were tert.-butanol-water  - 56 -  (70/35) and n-butanol-glacial acetic acid-water (40A0/50). (i)  Color reactions Nickel reagent  - The chromatograms, when dipped i n a 1%  nickel chloride solution, revealed no spots even after drying, except f o r the BAL standard which gave a chocolate brown spot with an Rf value of 0.91. Nitroprusside reagent  - When the chromatogram was dipped  i n the nitroprusside solution, no spots were obtained except for the BAL standard.  On drying, however, several spots appeared. These spots a l l  showed considerable streaking so that i t was difficult to determine i f any Other spots were masked by these t a i l s .  A well defined spot, with an Rf  value between 0,25 - 0.29, was present i n a l l the urine samples. This compound was also present i n the pre-injection urine.  When the f i l t e r  paper, containing this spot was cut out from the chromatogram and placed under the Geiger counter, i t was found to contain radioactive material. Another well defined spot was found having an  value between 0.49 - 0.54.  This compound was found In a l l the pre-injection urine samples but dissappeared from the urine after the injection of the BAL.  After 24 hours  this compound reappeared i n the urine. No radioactivity was found i n this spot. Bromo-cresol green  -  When the chromatograms were sprayed  with a 0,04$ solution of bromo-cresol green (50), well defined yellow acidic spots and blue basic spots were obtained. A- faint yellow spot (Rf = 0.10) was present on allthe chromatograms of the urine samples.  - 57 This spot was found to contain considerable radioactivity.  A compound,  giving a blue spot (Rf = 0.15 - 0.17), was found i n a l l the urine samples but contained no radioactivity.  Two other acidic compounds (Rf = 0.24 -  0.28 & Rj = 0«43 - 0*48) were also present i n a l l urine samples and were found to contain some radioactive material. An additional acidic compound (Rf = 0.67 -0.70) was found only i n the pre-injection samples although the urine sample of group D contained a very slight amount i n the 0 - 3 hour period after injection. Benzidine reagent  - After the chromatogram was sprayed  with benzidine reagent (36), a large brown spot appeared i n the 0 - 3 and 3 - 6 hour urines. This compound was not present i n the pre-injection urine and 12 hours after the injection i t had again disappeared.  However,  because both glucose and glucuronic acid gave brown spots with Rf values, very close to the Rf value of this compound, no conclusions could be made as to the identity.  The n-butanol-acetic acid-water solvent of Partridge  (51) was used,therefore, i n an attempt to determine the nature of this compound and the chromatograms were sprayed with either the benzidine reagent or ammoniacal silver nitrate (50). With this solvent, glucose was found to give a golden-brown spot (Rf = 0.18) when the paper was sprayed with the benzidine reagent, while glucuronic acid gave a red-brown spot (Rf = 0.33) and a smaller brown spot (R^ = 0.13). The unknown substance present i n the 0 - 3 and 3 - 6 hour urine samples was found to give a golden-brown spot with an Rf value identical to that of glucose. A very small spot with an Rf value of 0.33 was found i n a l l the urine samples. It would appear from these results that there was no increase i n the  - 58 excretion of glucuronic acid after the injection of BAL since the preinjection urines contained as much of this compound (Rf =0.33) as was found i n the post-injection urine. Similar results were obtained using the ammoniacal silver nitrate reagent. A Benedict's test was run on the pre-injection urines and the 0 - 3 hour sample. The pre-injection sample gave -a negative test whereas the 0 - 3 hour sample gave a strong positive test.  It was evident, therefore,  that there was present i n the post-injection urine a very large amount of a reducing carbohydrate which, from chromatographic studies, appeared to be glucose. (ii)  Radiochromatograms The results obtained from the radiochromatograms are shown  i n Figures 10 and 11. The presence of five urinary metabolites was i n dicated and the possibility of a sixth compound was also observed.  The  number of metabolites and the relative Rf ranges agreed with the results obtained i n Experiment 2. Since the solvents used i n the two experiments varied as to the amount of water, the R^ range was slightly different. The R- range of each compound was as followsj Compound 1  0.07 - 0.10  Compound 2  %  0.25 - 0.30  Compound 3  Rf  0.45 - 0.50  Compound 4  Rf  0.60 - 0.65  Compound 5  Rf  0.78 - 0.83  Compound 6  Rf  0.95 - 0.98  (26)  4  20  20  20  6P. C 0 - 3 HRS.  GP. C 3 - 6 HRS.  GP C 6-12 HRS  z  2  a.10 u  o  1.0  Ml  2  0  20  o  i.o  OS  20  BAL  GP. C 24-48 HRS.  BAL STANOARD  Z  2  *I0  *20  6.  o  6  1.0  OS  40  GR C 12-24 HRS.  o: 10  lU  10  I  o.s  (24) O  6  J1  (2) O.S  1.0  J  CtL 0  O.S R„  1.0  0  0.5 R .  1.0  Figure 10, Radioehromatograms of urine from group C rats injected intraperitoneally with 35BAL. Solvent tert. butanol-water (70/35), The numerals above the peaks indicate the number of the compound. Bracketed numerals indicate doubtful peaks. S  0  0.S  1.0  0  0.S  1.0  0  O.S  1.0  Figure 11. Radiochromatograms of urine from group D rats injected intraperitoneally with S-°~BAL. Solvent tert. butanol-water (70/35). The numerals above the peaks indicate the number of the compound. Bracketed numerals indicate doubtful peaks.  - 61 As i n Experiment 2, compound 1 was present i n largest amounts i n the 0 - 3 hour urine sample.  The amount of this compound slowly decreased so that  after 24 hours i t could just be detected.  Compound 3 was present i n most  samples i n substantial amounts, although after 24 hours the amount present could just be detected.  As i n Experiment 2 the major metabolite appeared TABLE XT?  Radioactive metabolites of BAL present i n the urine of groups C and D Urine sample Compound  Rf range  Time period  0-3 hr. 3-6 hr. 6-12 hr. 12- 24 hr. 24-48 hr.  Group  C  C  D  C  D  C  D  C  D  +  +  +  +  -  +  -  +?  1  0-07-0.10  + .+  +  2  0.25-0.30  +  -  +  +  +  -  +?  +  3  0.45-0.59  +  +  +  +  +  +  +  +  4  0.60-0.65  +  +  +  +  +  +  +  5  0.78-0.83  +  +  +  +  -  6  0.95-0.93  -  -  -  -  -  - -  + +  +  +  - -  +?  -  -  + - Compound present i n urine - - Compound absent i n urine +? - Presence of compound indicated, but peak not large enough to be classified as + to be compound 4. This compound was present i n large amounts even i n the last 24 hour sample.  Compound 5 was present i n most of the urine samples  as was compound 2. Compound 6 was present only i n very small amounts so that i t was extremely d i f f i c u l t to identify i t .  A summary of the radio-  0  - 62 active metabolites present i n the urine is given i n Table XIV, (iii)  Urinary extraction studies In order to obtain further information as to the chemical nature  of the various urinary metabolites of BAL, a method was set up f o r the extraction of the urine with various organic solvents.  Three ml. of the 3 -  6 hour urine sample of group D were adjusted to pH 3.0 and extracted three times with 2 ml, portions of peroxide-free ether.  The extracted urine was  further extracted with three 2 ml. portions of freshly d i s t i l l e d n-butanol saturated with water. The method is'schematically represented as follows: 3 ml. urine (pH  3.0)  peroxide-free ether  ether extract (I)  extracted urine n-butanol I  n-butanol extract (ID  extracted urine (HI)  Each fraction was then chromatographed and the chromatograms treated with bromo-cresol green (solvent tert-butanol/H^O) and ammoniacal silver nitrate (solvent n-butanol/acetic acid/B^O).  Radioehromatograms were also  run and the results obtained were compared with those obtained from a sample of the pre-extracted urine. The results obtained from the radioehromatograms are shown i n  - 63 Figure 12. The only compound that was extracted to any extent by the ether was compound 2. Compounds 1, 3, 4 and 5 were completely absent i n the ether extract.  However, considerable radioactivity waspresent i n the Rf. range  on either side of compound 4, indicating that this compound may be composed of several components. A l l of compound 5 was extracted i n the n-butanol solvent as was the majority of compound 3. Small amounts of 2 and 4 were also present i n the n-butanol extract.  Compound 1 was found entirely i n  the extracted urine as was the majority of compound 4. The chromatogram sprayed with bromo-cresol green gave the following results: Rf of spot Color of spot  0.08-0.10  0.20-0.24  0.40-0.44  yellow  yellow  yellow  Pre-extraction urine  +  +  +  Ether extract  -  -  -  n-butanol extract  -  +  +  Extracted urine  +  +  +  Each of these spots i n the pre-extracted urine was found to contain radioactivity and corresponded very closely to the Rf range given for compounds 1, 2 and 3. However, i t should be noted that although compound 2 was extracted by the ether solution, no yellow spot was obtained when the ether chromatogram was sprayed with bromo-cresol green.  This would seem to  indicate that the yellow acidic spot with an Rf range 0.20 - 0.24 was not due to compound 2.  64  20  20  ETHER  PRE-EXTRACTION  EXTRACT  URINE  I  S  aJlO  Jl  Z  CLIO u  3  [Lr  LT  1  r  0.5  0.5  1.0  1.0  20  20  EXTRACTED  N-BUTANOL EXTRACT  URINE  n  m  S  *I0  "lO u  6  2 0  3 0.5  1.0  Jl  Xn. 0.5  i.o  R,  Figure 12. Extraction studies on the 3 .-'6 hour urine sample of group D. The urine was adjusted to pH 3.0 and extracted f i r s t with ether and then with n-butanol saturated with water. Radioehromatograms were then run on each solution.  c  - 65 The chromatograms sprayed with ammoniacal silver nitrate showed that almost a l l the reducing compound (Rf = 0,18) remained i n the extracted urine.  A very small amount was found i n the n-butanol extract. A l l other  spots normally present on chromatograms of this urine sample were found i n the extracted urine only, except for a very small amount which was found i n the n-butanol extract. A compound which gave a dark brown spot (Rf = 0.88), and which was present i n this post-injection urine sample, was completely removed into the ether extract. (iv) ft -glucuronidase studies It had been reported by the Oxford group (17) that the injection of BAL into rats gave rise to a large increase i n the glucuronic acid excretion, and that the possibility arose that BAL or one of i t s metabolites might be excreted as the glucuronide i n the urine. From the chromatographic studies described above, no indication of an increase i n the glucuronic acid excretion could be found.  I f , however, the glucuronic acid were present  i n the urine as the glucuronide i t would not be detected on the f i l t e r paper chromatogram. Therefore, i n order to determine whether one of the metabolites of BAL was present as the glucuronide, the following experiment was carried out.  A portion of the 3 - 6 hour urine sample of group D was adjusted to  pH 4.5 and to 1 ml. of this solution was added 5 mg. of calf spleen glucuronidase*.  The remainder of the urine was used as a control.  £  -  After 8  * Kindly supplied by Mr. Jack Phinney of Shaughnessy Hospital's Biochemistry Laboratory.  - 66 hours at 37° C, the urine samples were chromatographed and the chromatograms treated with bromo-cresol green and ammoniacal silver nitrate.  Radio-  ehromatograms were also run on both urine samples. The results obtained from the radioehromatograms 13.  are shown i n Figure  No indication of any change could be found i n the urine sample In-  cubated with ft -glucuronidase. The spots which appeared on the chromatogram sprayed with bromo-cresol green showed no change i n position from those found i n the control urine, the three yellow acidic spots (Rf = 0.08; 0.20; 0.43) being found i n both samples. After spraying the chromatograms with ammoniacal silver nitrate no spots not already present i n the control were found i n the urine containing the /3 -glucuronidase. Also, no increase i n the amount of free glucuronic acid was found i n the urine after treatment with  P-glucuronidase.  (v) Acid hydrolysis In order to determine whether any of the metabolites were labile to acid the following experiment was carried out.  A sample of the  3-6  hour urine of group D was acidified to a pH less than llwith- concentrated hydrochloric acid.  The urine was heated i n a sand bath at 100° C. for  30 minutes, cooled and chromatographed. The chromatograms were then sprayed with bromo-cresol green.  Radioehromatograms of this urine sample were also  run. The results obtained from the radioehromatograms 14.  are shown i n Figure  It was found that compound 3 had completely disappeared and that  4 (24)  20  20  B-6LUCUR0NIDASE  if  2  °i IOJ l u  CONTROL  2  o: 10  6  5  n 5  Lr  l_n 0.5  1.0  0.5  Figure 13. ^ -glucuronidase action on group D urine. Radioehromatograms of the 3 - 6 hour urine sample of group D before and after -glucuronidase action.  1.0  5 (27) 20  20  CONTROL  ACID HYDROLYSIS  2  a| 10  6  u  1  2 0.5  1  :I0  I  1.0  cn  2  CD  iho  0.5  Figure 14. The effect of acid hydrolysis on the various radioactive metabolites present i n the urine after an injection of S 5_BAL. The 3 - 6 hour urine sample from group D was acidified to a pH less than one and heated to 100° C for 30 minutes. Chromatograms were then run on the hydrolysed sample and the control. 3  1.0  - 69 compound 5 was present i n much greater amounts. The results obtained from the chromatogram sprayed with bromo-cresol green showed the presence of a new acidic compound (Rf. = 0.76) i n addition to the three acidic compounds which were normally present i n this urine sample.  It was found, however,  that the yellow spot (Rj> = 0.43) was no longer radioactive, while the new acidic spot contained considerable radioactivity.  It would appear, there-  fore, that the acidic spot (Rf = 0.43) was not due to compound'3. ( i) v  Characterization of compound 1 as S0^~ Since urinary sulphur analysis showed that a small amount of  the injected S 5 was present i n the urine as s35o^~, the following experiment 3  was carried out to determine which of the urinary metabolites was due to r  s55-Oj~. A sample of the 3 - 6  hour urine of group D was treated with a few  drops of 5% barium chloride solution.  The precipitate formed was centrifuged  and the supernatent chromatographed. A chromatogram of an authentic S-^o^"" solution was also run.  The results obtained are given i n Figure 15.  On addition of the barium chloride solution, Compound 1 was completely removed from the urine. It was also found that the Rf. value of the authentic s35o = was identical with that of compound 1. 4  This evidence, taken i n con-  junction with other result s described above where i t was found that compound 1 was insoluble i n organic solvents and gave a faint yellow acidic spot with bromo-cresol green, would indicate that compound 1 was present as s35o^~. b)  Sulphur analysis  Figure 15. -Characterization of compound 1 as S^O* . The figure on the l e f t i s the radiochromatogram of the 3 - 6 hour urine sample of group D before addition of BaClp. The centre diagram shows the radiochromatogram obtained after precipitation of BaSO.. The results obtained with an authentic sample of S350 ~ is shown i n the diagram on the right. 4  - 71  (i)  -  Total sulphur The total sulphur content of each urine sample was determined i n  duplicate by the method used i n Experiment 2. given i n Table XV. total  The results obtained are  The rates of excretion of the total sulphur and of the  -sulphur during the various time intervals are shown i n Figure 1 6 .  The rates of excretion of the total sulphur were compared with the rates of excretion on the three days prior to injection of the BAL.  The amount of  injected S ^ excreted i n the urine during the various time periods of the 3  experiment i s given i n Figure 17. (ii)  Inorganic sulphate .The inorganic sulphate content of each urine sample was de-  termined i n duplicate using the method described previously. In the group C determinations, urine samples for the three days prior to injection were used as controls. The results obtained are shown i n Table XVT.  The rates  of excretion of the inorganic sulphate and the inorganic s35-sulphate are given i n Figure 1 8 . (iii)  Total sulphate The total sulphate content of each urine sample i n group C was  determined i n duplicate using the method described previously. Since there was insufficient urine i n the group D urine samples to run duplicates no analysis was attempted. in Table XVII.  The results obtained with group C urine are shown  The rates of excretion of the total sulphate and the total  S35_sulphate are shown i n Figure 19 <»  TABLE XV Total sulphur and total S 5-sulphur i n rat urine after intraperitoneal injection of 242,000 c.p.m. of S BAL/group of three rats 3  Group Time period after i n jection (nr. )  C  Volume of urine excreted (ml.)  Total S (mg.)/ml. urine  Total S excreted (mg.)  Total S (nig.)/  hr.  Total S (c.p.m,)/ ml. urine 3 5  Total S excreted (c.p.m.)  Total S (c.p.m.)/ hr.  3 5  3 5  0-3  3.2  2.7  8.6  2.9  13,745  43,880  14,630  3-6  3.9  1.7  6.6  2.2  8,125  31,690  10,560  6-12  2.1  3.6  7.6  1.3  7,140  14,990  2,500  12 - 24  11.8  1.0  11.8  1.0  2,802  33,060  2,740  24 - 48  8.0  1.5  12.0  0.5  1.302  10,420  435  Pre-i.njection urine Day 1 11.25 hr. Day 2 12.0 hr. Day 3 13.0 hr.  D  33  Total 3.8 3.6 3.5  3.4 2.8 3.5  0-3  3.6  1.4  12.3 5.0  3-6  7.0  1.5  6-12  4.7  12 - 24  24 - 48 Pre-injection urine Day 1 11.25 hr. Day 2 12.0 hr. Day 3 13.0 hr.  12*9"  10.1  134,040  1.1 0.8 0.9 1177  8,950  32,220  10,740  10.5  3.5  6,800  47,600  15,870  1.4  6.6  1.1  5,120  24,060  4,010  13.0  0.9  11.7  1.0  2,282  29,670  2,490  7.2  2.1  15.1  0.6  1,197 Total  8,620 142,170  360  4.6 3.8 6.7  2.2 2.3 2.6  10.1 8.7 17.4  0.9 0.7 1.3  - 73 -  Figure 16. Rate of excretion of t o t a l sulphur and total S ^sulphur at various time intervals following intraperitoneal i n jection of S 5-BAL. The rate of total sulphur excretion f o r the three days prior to injection i s also shown. 3  3  TABLE XVI Inorganic sulphate and inorganic S -sulphate i n rat urine after intraperitoneal injection of 242,000 c.p.m. of S35-BAL/group of three rats 33  Group Time period after i n jection (hr. )  C  Volume of urine excreted (ml. )  Inorganic S0 (mg.)/ml. urine  4  Total i n organic SO4 excreted (mg. )  Inorganic Inorganic S0 "(mg.)/ s35o4" hr. (c.p.m.)/ ml. urine 4  35  4  Inorganic  s V 3  (c.p.m.) / hr.  0-3  3.2  0.45  1.44  0.48  1,720  3-6  3.9  0.34  1.33  0.44  610  2,380  790  6-12  2.1  0.86  1.80  0.30  555  1,170  190  12 - 24  11.8  0.57  6.70  0.56  257  3,030  250  24 - 48  8.0  1.25  0.42  207  1,660  70  10.0  Total Pre-Injected urine Day 1 11.25 hr. Day 2 12.0 hr. Day 3 13.0 hr.  D  Total i n organic S 0 ~excreted (c.n.m.) 5,500  1,830  13,740  3.8 3.6 3.5  1.00 0.85 1.00  3.80 3.06 3.50  0.34 0.25 0.27  0-3  3.6  0.46  1.66  0.55  2,520  9,060  3,020  3-6  7.0  0.17  1.19  0.40  1,350  9,450  3,150  6 - 12  4.7  0.28  1.32  0.22  655  3,080  510  12 - 24  13.0  0.34  4.42  0.37  325  4,220  350  24-48  7.2  2.00  0,60  223  1.600  67  14.4  •  total  27.410  - 76 -  0.6  GR C  INORGANIC  ^3000  GP. C  i  SULPHATE  ti  M G./HR. 0.4  1-2000 < x a.  bl  _i . 3 (0  gl  to  1000  <  o o z  DAY DAY I 2  n n  0 - 3 3 - 6 6-12 12-24 24-48 TIME PERIOD HRS.  DAY " 0 - 3 3--6 6-12 12-2424-46 T I ME 3 I PERIOD HRS. INJECTION  3000  0.6  GP. D  «P. D 0£  6  INORGANIC  £2000  OA  <  X  SULPHATE  3  MG. / H R .  co to 1000  Si  0.2  < ©  o  0 - 3 3 - 6 6-12 12-24 24-48 TIME PERIOD HRS.  n  -3 3 - 6 6-12 12-242448 TIME PERIOO HRS.  Figure 18. Rate of excretion of inorganic sulphate and inorganic s"_sulphate at various time intervals following intraperitoneal injection of S -BAL. The rate of inorganic sulphate excretion for the three days prior to injection i s also shown for group C urine samples.  TABLE XVII Total sulphate and total  Group  C  Time period after i n jection (hr.)  -sulphate i n rat urine after intraperitoneal injection of 242,000 c.p.m. of BAL/group of three rats  Volume excreted urine (ml.)  Total S0 Total S0A (mg.)/ml. collected during time urine period(mg.) 4  Total S0 (mg.)/ hr. 4  Total s35o (c.p.m.)/ ^ ml, urine  -  Total S35o collected during time period (c.p.m.)  4  Total s35o (c.p.m.)/ hr.  0-3  3.2  -  ' -  -  3-6  3.9  0.40  1.56  0.52  635  2,480  830.  6-12  2.1  0.57  1.20  0.20  605  1,270  210  12 - 24  11.8  0.57  6.70  0.56  280  3,300  270  24 - 48  8.0  1.25  0.42  240  1,920  80  10.0  -  -  4  ( - 78 -  Figure 19. Rate of excretion of total sulphate and total S^_ sulphate at various time intervals following intraperitoneal injection of S35_BAL. Group C only i s shown since there was insufficient urine i n group D samples to carry out analysis. 3  DISCUSSION In the experimental work described, a study was made as to the nature of the urinary metabolites of BAL i n the rat after injection of S"^-BAL.  A detailed study was also carried out concerning the amount of  S^5 present i n the urinary sulphur fractions at various periods of time after injection of S25-BAL. It was reported by Peters and co-workers (31) that, following an intraperitoneal injection of S^-BKL  into the rat, approximately 45$ of  the injected S-> was excreted i n the urine within 24 hours while 50$ was 3  Sjjnp n and Young (32) reported that over 70$  excreted within 48 hours.  SO  35 of the injected S  J  was excreted i n the f i r s t 24 hours after intramuscular  injection of s35_BAL  Similar results were obtained i n the experiments  e  described herein.  In Experiment 2 approximately 50$ of the injected S ^ 3  was excreted during the f i r s t 6 hours after intraperitoneal injection of S ^-BAL. 3  After 48 hours group A had excreted 69$ of the injected S ^ 5  while group B had excreted 80$. In Experiment 3 the amounts of S ^ ex3  creted by groups C and D were somewhat lower. 35 of the injected Or-' had been excreted. i n i t i a l excretion of S 5 3  w  a  s  After 48 hours 55 - 60$  However, i n a l l groups a rapid  recorded i n the f i r s t 6 hours.  The amount  35 of £\  J  excreted slowly decreased so that i n the last 24 hours of the ex-  periment there was very l i t t l e excretion of S ^ (Figures 8 and 17). It would appear, therefore, that there i s some retention of the injected Since no studies were carried out on the carcasses of the injected 3*5 animals the site of retention of the injected S known.  J  i n these animals i s not  - 80 The maximum rate of excretion of total s35-sulphur was found to take place during the f i r s t 6 hours (Figures 7 and 16).  In groups A, and C the  maximum rate was observed during the 0 - 3 hour period while with group B and D the maximum rate of excretion was obs erved. during the 3 - 6 hour period.  This was followed by a very rapid decrease i n the'rate of ex-  cretion of S . 55  A parallel increase i n the rate of excretion of total sulphur was also noted (Figures 7 and 16).  The rate of sulphur excretion rose  from the pre-injection level of approximately 1 mg./hr. to a level of 3 mg./hr. during the 0 - 6 hour period. The rate of excretion of total sulphur returned to pre-injection levels within 12 hours. In Experiment 2, 4 $ of the S  3 3 W  a s found to be excreted as sulphate.  In Experiment 3, however, the percentage of  excreted as sulphate was  found to "be 10$ for group C and 19$ for group D. The values obtained i n Experiment 3 are considerably higher than those obtained i n Experiment 2 and are also higher than the value of 8$ obtained by Peters et a l (31) and 3$ reported by Simpson and Young (32).  Since a l l the S  3 3  present i n BAL  is i n the form of neutral sulphur, any s35„ iphate present i n the urine su  would have to arise from the oxidation of the neutral S 5_ iphur. 3  su  One  would expect, therefore, that the longer the S ^-sulphur remains i n the 3  body, the greater the possibility of oxidation to S35_sulphate.  If this  were the case the ratio of S35-sulphate to neutral S35-sulphur should decrease with time. in Table XVIII.  This was found to occur i n groups A and B as i s shown  However i n group C there was an i n i t i a l low ratio during  81 the f i r s t 3 hours after BAL injection.  The ratio increased to a maxi-  mum i n the 3 - 6 hour period, and slowly decreased for the remainder of TABLE XVIII Ratio of S 5_3ulphate/neutral S 5_sulphur i n the urine of rats at various intervals of time after injection of S35_BAL 3  Time interval after injection (hours)  3  s35- ulphate/neutral s35_ ulphur S  Group A  S  Group B  Group C  Group D  0-3  0.025  0.025  0.143  0.390  3 - 6  0.042  0.022  0.081  0.248  6-12  0.057  0.067  0.085  0.147  12 - 24  0.065  0.082  0.100  0.166  24 - 48  0.135  0.151  0.190  0.228  the experiment i n a manner similar to groups A and B.  In group D a  very low ratio was found i n both the 0 - 3 and 3 - 6 hour urine samples. A maximum ratio was obtained, however, i n the 6 - 1 2 hour period and slowly decreased for the remainder of the experiment. It i s of interest to note that i n the 0 - 3 hour radioehromatograms of groups C and D and the 3 - 6 hour radiochromatogram of group D compound I, which was later identified as sulphate, was present i n very large amounts (Figures 10 and 11). The reason for this i n i t i a l rapid oxidation of neutral sulphur to sulphate i n groups C and D i s not known. The following facts, however, should be noted. The toxic effect of BAL on groups C and D was much greater than that on groups A and B. The fact that a l l the rats i n  - 82 groups C and D experienced quite severe convulsive seizures indicate that the normal metabolic pattern was severely upset.  When one considers the  inhibitory effect of BAL on certain cellular enzymes due to i t s effect on essential metal coenzymes (19) and i t s activating power on other enzyme systems (55), i t may be that the anomalies described above were due to abnormal metabolism of the injected BAL. The rate of S^-sulphate  excretion was found to reach a maximum  during the 3 - 6 hour period i n a l l groups except group C i n which case the maximum rate was obtained during the 0 - 3 hour period (Figures 9, 18 and 19).  The rate of excretion for a l l groups f e l l off rapidly, for the  remainder of the experiment. The excretion of total sulphate, however, did not parallel the excretion of total  sulphate.  Although there was an i n i t i a l increase  i n the total sulphate level, there was additional increase during the last 36 hours of the experiment. This latter increase could not be explained by the normal physiological variation i n sulphate excretion but may have been due to tissue damage at the site of injection.  Cuthbertson (56) has  shown that after injury there i s an increased sulphur excretion due to an increase i n the inorganic sulphate excretion. The residue obtained on acid hydrolysis of the urine i n Experiment 2 was found to contain a widely varying amount of S 5 (Table XIII). In 3  the 0 - 3 hour samples of both groups the amount of S 35 the total Sr^ excreted during this time period.  3 3  found was 25% of  However, i n the majority  - 83 of the other urine samples the amount of radioactivity present was so small that no measurements could be carried out.. Therefore, no conclusions could be made concerning the nature of the S"^ found i n this residue. It would appear, however, that the majority of the S-*5 i n this residue was due to neutral S ^-sulphur. 3  In Experiment 3 the amount of ethereal S^^-sulphate was only 0.5$ of the excreted S35.  One may conclude, therefore, that the amount of S35-  BAL converted to ethereal sulphate is very small.  Similar results were  obtained by Peters et a l (31) who found the S 5 content of ethereal sul3  phate was either zero or a very small fraction of the t o t a l . Stocken and Thompson (10) reported that a rapid increase i n the urinary t h i o l excretion occured within the f i r s t hour after BAL injection into the rabbit. Similar results, i n the experiments described herein, were obtained i n the rat. It was found that the maximum excretion of thiols as measured by the nitroprusside reaction, occured during the 3 - 6 hour 35 period following injection of S -BAL. The amount of t h i o l decreased during the 6 - 1 2 hour period so that after 12 hoars the urine once again gave a negative nitroprusside test. The chromatographic results indicated the presence of 6 possible metabolites.  There was, moreover, no indication of the presence of any  excreted BAL.  The absence of BAL i n the urine supports the results ob-  tained by Simpson and Young (32) who found that on extracting the postinjection urine of rats given s'^-BAL, no radioactive BAL could be detected.  84 Compound 1 was present i n largest amounts during the f i r s t 6 hours after injection of the S-*-BAL. This compound gave a faintly yellow 3  acid spot when the chromatogram sprayed with bromo-cresol green,and was found to be insoluble i n ether and n-butanol.  The addition of 5% barium  chloride solution to the urine caused this compound to precipitate out. Since i t was found to have the same  value as authentic S^O^ , i t was -  concluded that compound 1 was sulphate. Compound 2 was present i n small amounts i n the majority of the urine samples.  It was soluble to some extent i n ether and n-butanol.  Compound 3 was present i n considerable amounts i n almost a l l the urine samples. ether.  It was found to be soluble i n n-butanol but insoluble i n  On acid hydrolysis compound 3 was converted into compound 5.  This  would explain why compound 3 was absent i n the 3 - 6 hour urine sample of group A (Figure 4).  Instead, a very large amount of compound 5 was found.'  A direct relationship between the amounts of compound 3 and 5 found i n the urine was noted.  If the concentration of compound 5 was quite high then  compound 3 was present i n small amounts and i f the concentration of compound 3 was large then compound 5 was present i n small amounts. The nature of the group released on acid hydrolysis, however, i s not known. Compound 4 appeared to be the major metabolite.  It was found to  be insoluble i n ether and only slightly soluble i n n-butanol. Compound 5 was formed on acid hydrolysis of compound 3.  It i s not  known, therefore, whether this compound was excreted as such or whether i t  arose on standing i n the acidified urine. i n ether but very soluble i n n-butanol.  It was found to be insoluble This compound gave a greenish-  brown spot with the nickel chloride reagent and a positive nitroprusside test.  It would appear, therefore, that this compound contained a sulphydryl  group or groups.  It i s of interest to note that the dithiol isolated from  rabbit urine by Spray and co-workers (29) gave a greenish-brown colour with nickel salts.  They also found that the dithiol was much less soluble  i n benzene than was BAL. The fact that compound 5 was not extracted to any noticeable extent with ether but was removed entirely by n-butanol indicates that these compounds may be similar. Compound 6 occured only i n very small amounts i n the urine sample of groups C and D.  However, i n groups A and B the amount of this compound  present was sufficient to detect i t quite readily. Since compound 6 was not present i n the urine samples used for the extraction studies no results as to i t s solubility i n ether or n-butanol are available. Experiments were carried out to determine whether BAL, i f excreted i n the urine, might undergo oxidation to from one of the 6 metabolic products found i n the urine.  The results showed, however, that none of  these compounds arose from BAL oxidation i n urine.  Even after 48 hours  there was no indication of any new compounds being formed by oxidation of the S-^-BAL (Figure 6).  The fact that the BAL could no longer be detected  by colour reagents after 12 hours, even though the amount of BAL present was s t i l l quite large, emphasizes the point that a negative nitroprusside or nickel chloride test does not mean that the various compounds, detected  - 86 on the radioehromatograms, are not thiols.  It may simply mean that these  compounds are present i n such small amounts that they could not be detected by the colour forming reagents. Studies with P glucuronidase failed to indicate the presence of a glucuronide (Figure 13).  There was also no indication of an increased  glucuronic acid excretion after BAL injection.  These results differ from  those reported by Spray (30) i n which a marked increase i n urinary glucuronic acid excretion occured after injection of BAL. These results could not be substantiated i n the experiments described herein.  In both the pre-injection  and post-injection urine very small amounts of glucuronic acid was detected on the f i l t e r paper chromatograms. However, there was no increase i n the amount of glucuronic acid detected i n the post-injection urine even after acid hydrolysis and p glucuronidase action. From these results one can conclude that i n the experiments described herein thece was no increase in the excretion of glucuronic acid either i n the free form or as the glucuronide. The presence of large amounts of glucose, however, were indicated from the chromatographic studies. A strong positive Benedict*s test was also obtained on the post-injection urine.  Rats injected with propylene  glycol, which was used as the solvent for BAL i n the injections, showed no signs of glucosuria.  It was concluded, therefore, that the glucosuria  was due i n some manner to the injected BAL. As f a r as one can determine there has been no reference i n the literature to this phenomenon.  There  are reports, however, of increased blood sugar levels after BAL injection (57, 58).  The glucosuria, therefore, may be due to a hyperglycemic con-  dition i n the rats after BAL injection.  - 88SUMMARY 1.  Techniques have been established for the oxidation of sulphur i n organic material and the precipitation of the sulphate formed as benzidine sulphate. The methods can be used for measurement of S i n organic materials by determining the radioactivity of the benzidine sulphate.  Self absorption corrections have been determined f o r thin  samples of benzidine S -sulphate. 33  2.  Radioactive 2:3-dimercaptopropanol  (BAL) was prepared by reacting  NaS H with 2:3-dibromopropanol i n a closed system at 60° C. The 35  NaS H,was prepared from BaS 33  33  obtained by the reduction of BaS 5o 3  4  with sugar charcoal at 1000° C. The S -BAL was characterized bys 33  a)  determination of i t s sulphydryl content.  b)  preparation of the following crystalline derivativess i) ii)  c)  2:2-dimethyl-4-hydroxymethyl-l:3-dithiolan 2-phenyl-4-hydroxymethyl-ls3-dithiolan.  chromatographic behavior.  The specific activity of the S -BAL was determined from S 5 analysis 33  3  on the BAL and on the 2-phenyl-4-hydroxymethyl-l:3-dithiolan« 3.  A method has been developed f o r the precipitation and measurement of S  3 3  i n the inorganic and ethereal sulphate fractions of urine. This  procedure was based on the methods of Owen (44) and McKittrick and Schmidt (45).  - 89 4.  A method has been developed for the detection of micro amounts of BAL by f i l t e r paper chromatography. The solvent used was tertiary butanolwater (70/35j V/V)  a n  d the position of the BAL on the f i l t e r paper was  located by dipping the f i l t e r paper i n a 1% aqueous solution of nickelous chloride, which formed a chocolate brown colour with BAL. as 8yug. of BAL could be detected by this reagent.  As l i t t l e  Monothiols, such  as cysteine, gave a green spot with this reagent and could only be detected i n much larger amounts. 5.  A technique has been devised for the radiochromatographic of the various metabolites of  detection  -BAL present i n the urine. A strip,  1 1/8" wide, containing the path followed by the urine spot, was cut from the chromatogram. This strip was then cut into 1 cm. sections and each section was counted under the Geiger-Muller tube.  The  counts per minute were plotted against the distance the particular section was from the point of application of the original spot. 6.  The metabolism of S 5_BAL was studied i n the Wistar rat. The radio3  active compound was injected intraperitoneally and a detailed study of the S ^ content of the various urinary sulphur fractions was 3  carried out at various time intervals after the injection. mum rate of  The maxi-  excretion took place during the f i r s t 6 hours f o l 35  lowing administration of the S decrease i n  -BAL.  This was followed by a rapid  excretion for the remainder of the experiment. There  was a corresponding increase i n the anount of neutral sulphur and inorganic sulphate present i n the urine after BAL injection.  The  90 neutral sulphur level returned to normal within 12 hours whereas the inorganic sulphate level remained abnormally high throughout the experiment.  Approximately 4 - 19$ of the excreted S  3 3  was present as  inorganic sulphate while less than 0.5$ was excreted as ethereal sulphate. 7.  Six possible metabolic products, none of which i s BAL, have been shown to be excreted i n the post-injection urine. These compounds were detected by means of the radiochromatographic technique and were found to have the following Rj values when a tertiary butanol-water solvent (70/35) was used:  Compound 1  0.07 - 0.10  Compound 2  0.25 - 0.30  Compound 3  0.45 - 0.50  Compound 4  0.60 - 0.65  Compound 5  0.78 - 0.83  Compound 6  0.95 - 0.98  Compound 1 was characterized as inorganic sulphate. The others, as yet unidentified, appear, from extraction studies, to be organic i n nature and may be conjugates or oxidation products of BAL. Compound 3 was found to be converted to compound 5 by acid hydrolysis. Compound 5 appeared to be similar to the dithiol isolated by Spray et a l (29) from rabbit's urine.  - 91 8,  No evidence has been found to support the theory that BAL might be excreted as the glucuronide.  Also, no increase i n glucuronic acid  excretion could be detected after BAL injection.  The presence of a  large amount of glucose, however, has been demonstrated i n the urine for the f i r s t 6 hours after injection of BAL.  -  92 -  BIBLIOGRAPHY  1.  E h r l i c h , P., Ber., 42, 17 (1909).  2.  Voegtlin, C , Physiol. Rev., 5_, 63 (1925).  3.  Rosenthal, S.M., U.S. Publ. H l t h . Rep., 47, 241 (1932).  4.  Cohen, A., King, H. and Strangeways,,W.I., J . Chem. S o c , 3043 (1931).  5.  Thompson, R.H.S., Biochem. J . , 40, 525 (1946).  6.  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Simpson, S.D., Zcarsky, S.H. and Young, L., Report to Directorate of Chemical Warfare, Canada, C.E., 44, Report No. 12 (1944).  43.  Hawk, P.B., Oser, B.E; and Summerson, W.H., Practical Physiol. Chem., Blakiston*'Co., Toronto (1947).  44.  Owen, C.E., Biochem. J., 30, 352 (1936).  45.  McKittrick, D.S. and Schmidt, C.L.A., Arch. Biochem., 6_, 411 (1945).  46.  Fiske, C.H., J. Biol. Chem., 47, 59 (1921).  47.  Martin, A.J.P. and Synge, R.L.M., Biochem. J., 35, 1358 (1941).  48.  Toennies, G. and Kolb, J.J., Anal. Chem., 23_, 823 (1951).  49.  Miller, H. and Kraemer, D.M., Anal. Chem., 24, 1371 (1952).  50.  Berry, H.K., Sutton, H.E., Cain, L. and Berry, J.S., Univ. Texas Publ., 5109, 22 (1951).  51.  Partridge, S.M«, Biochem. J., 42, 238 (1948).  52.  Tomarelli, R.M. and Florey, K., Science, 107, 630 (1948).  53.  Taylor, J.D., Richard, R.K. and Tabern/D.L., J. Pharm. Exp. Therap., 104, 93 (1952).  54.  McDonald, F.F., Brit. J. Pharmacol., 3_, 116 (1948).  55.  Nachmansohn, D., i n Advances i n enzymology, Vol. XI, Interscience, New York (1951).  56.  Cuthbertson, D.P., Biochem. J., 25_, 236 (1931).  57.  Graham, J.D.P., and Hood, J., Brit. J. Pharmacol., 3_, 84 (1948).  58.  Hulsman, T.H.J., Lammers, W. and Siderius, P., Acta Physiol. Pharmacol. Neerl., 1, 184 (1950).  

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