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Investigation on the biosynthesis and the chemosynthesis of glucuronic acid Ashford, Walter Rutledge 1941

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L^bft7 • n«< /? v  INVESTIGATIONS ON T H E BIOSYNTHESIS  AND T H E CHEMOSYNTHESIS OJF GLUOI3RONIO A3 ID.  BY  WALTER R. ASHFORD  ...A T H E S I S SUBMITTED I N P A R T I A L J D L E I L M E N T OP T H E REQUIREMENTS EOR THE DEGREE OJF MASTER OF ARTS I N T H E DEPARTMENT OE CHEMISTRY.  T H E U N I V E R S I T Y OE B R I T I S H COLUMBIA. APRIL,  1941.  I*  (i) *  !  ' 'TABLE OF .CONTENTS  TOPIC  PAGE  ACKNOICLEDGMENIS  (ii)  •: IHTRGDUGTION  . • 1  PHYSIOLOGICAL IMPORTANCE OF GLUCURONIC ACID  ,  : REVIEW OF METHODS OF SYNTHESIS (i) Biosynthesis  IS  ( i i ) Chemical methods of synthesis  15  PROPERTIES OF GLUCURONIC ACm-AND ITS COMPOUNDS  17  , EXPERIMENTAL WORK (a) Biosynthesis  19  (b) Chemical synthesis  25  (i) Oxidizing agents ( i i ) The effect of pressure  27 33  t  ( i i i ) The effect of temperature  34  (iv) The effect of P"  34  (v) Catalysts and t h e i r effects  37  (vi) S p e c i f i c i t y of naphthoresorcinol test ( v i i ) Methods of separation , CONCLUSIONS AND DISCUSSION BIBLIOGRAPHY  39 41 43  following 45.  (ii)  ACKNOWLEDGMENTS  I wish to express my thanks to Dr. R. H. Clark, under whose guidance this work was conducted, f o r h i s continued helpful and stimulating  interest, and for his many-  suggestions.  Thanks are also extended to Mr. William Barclay and Mr. Gordon Hewitt, i n conjunction with whom the work was done, to Mr. J . A. McCarter f o r valuable assistance and to Mr. G. Mathias and the Department of Bacteriolog for t h e i r active  co-operation.  INVESTIGATIONS ON THE BIOSYNTHESIS AND  INTRODUCTION  THE CHEMOSYNTKESIS OP GLUCURONIC ACID  ;  Glucuronic acid has always been of experimental value from a physiol o g i c a l point of view, but recently has taken on a new importance as a result of the discovery 1) of i t s function as a detoxification mechanism i n the animal body 2) of i t s possible role i n intermediary metabolism and 3) of i t s use i n the preparation of syruthetic antigens. To investigate these phases of the physiological chemistry of glucuronic acid, i t i s necessary to have a means of producing the substance i n pure form, so that large-scale experimentation may be carried out*  The  first  objective was therefore to devise a method, preferably chemical, by which glucuronic acid could be r e a d i l y obtained i n a pure state.  The physio-  l o g i c a l aspects of the problem have not been attacked as yet, but i t i s hoped that this more important phase of the problem w i l l be studied i n the near future.  In the following paper are given the results obtained from  the attempt to synthesize glucuronic acid from such compounds of glucose as the oxime and the °c methyiglucoside, I.  PHYSIOLOGICAL IMPORTANCE OF GLUCURONIC ACID.  To show the necessity of obtaining a satisfactory method of making available a ready synthesis of glucuronic acid, for biochemical i n v e s t i gation, a short account of the physiological importance of the acid w i l l be given. Glucuronic acid was shown some years ago to be present i n small amounts i n blood", l i v e r and urine, where i t exists i n the free or combined  form.  In 1932 Quick ( l ) showed that the animal organism can synthesize  glucuronic acid at the rate of one gram per hour, which finding led him to suggest that glucuronic acid may play a part i n the metabolism of carbohydrates or at least has some important physiological functions,  (i) As a detoxification mechanism  9  During the digestive process, certain nocuous substances are produced i n the intestine. tissues.  Others may r e s u l t from metabolic processes i n the  For, example, phenol may be produced from the breakdown of tyro-  sine and indole from the destruction of the tryptophane molecule.  Some  of these products are conjugated by glucuronic acid to form glucuronides i n which form theyeare excreted from the body.  t  In t h i s respect, glucuronic  acid seems to have a great a f f i n i t y f o r hydroxyl groups.  Such substances  as phenol and borneol are conjugated e a s i l y but terpenes are oxidized to •corresponding alcohols and then conjugated.  The action with hydroxyl  groups i s not s p e c i f i c as benzoic acid and " a s p i r i n " are also readily conjugated. It i s of interest to note the types of conjugation which occur. They are  l ) of the glucoside "ether" type such as that found i n d-bornyl  glucuronide, resulting i n a compound which i s non-reducing,  2) of the  glucoside "ester" type such as benzoyl glucuronide which retains i t s reducing properties and  3) the type containing two glucuronic acid molecules  one attached to a non-sugar residue by a glucoside "ether" linkage and the other attached by an "ester" linkage.  An example of this l a t t e r type  i s p-hydroxy benzoic diglucuronic acid.  ( i i ) Formation i n the body» Suggestions have been put forward to explain the formation of glucuronic acid i n the body.  Fischer (2) suggested two p o s s i b i l i t i e s to explain the  presence of glucuronic acid i n the body.  Glucuronic acid may be present  naturally, or may be formed by some mechanism i n the body and theaa conjugated with toxic substances introduced into or produced i n the body. A second p o s s i b i l i t y suggested, was that toxic substances unite with glucose to form glucosides which are then oxidized to form glucuronides.  The second  p o s s i b i l i t y has been shown to be untenable as shown by Pryde and Williams (3) who fed phenyl °c and & glucosides to animals and found that they were not converted into the corresponding glucuronides. Instead, they underwent hydrolysis and the liberated phenol was detoxicated and excreted as an ethereal sulphate.  Further, i t i s known that glucuronic acid i s not a normal oxi-  dation product of glucose metabolism.  Quick (4) has suggested that glucuronic  acid i s derived from body protein, basing h i s conclusions upon his findings that i n the diabetic animal, glucuronic acid i s produced from that portion of the protein molecule which would otherwise have gone to glucose. He believes that glucose and glucuronic acid have the same precursor i n the body.  In 1932 Quick (5) suggested that glucuronic acid i s probably syn-  thesized from short chain carbohydrate der ivatives.  This suggestion has  been expanded and proved with some degree of certainty by Lipschitz and Bueding (6) who by very careful experimentation found that the percentage of glucuronic acid synthesized i n v i t r o by surviving l i v e r s l i c e s was i n creased several hundredfold by the addition of dihydroxy acetone, pyruvic acid or l a c t i c acid to the medium.  They suggested that the oarbonyl group  of a t r i o s e probably conjugates with an alcohol and the resulting trioside i s b u i l t up to the conjugated acid.  ( i i i ) Factors influencing the production of glucuronic acid. The v e l o c i t y of production depends upon the nature of the substance to be conjugated.  I f a hydroxy! group has to be introduced by c e l l metabolism,  as i s the case with camphor, then the v e l o c i t y of production of the conjugated product i s slow.  Quick (5) has shown that i n s u l i n can markedly  increase, while acetoacetic acid as well as l a c t i c acid, decidedly suppress the output of glucuronic acid. ive  L i p s c h i t z and Bueding (6) have made exhaust-  tests on pure chemical substances which increase or decrease the pros  ducti'on of glucuronic acid i n surviving tissue s l i c e s .  They found that the  production of glucuronides was an oxidative process involving oxygen and that the oxidation process was catalyzed by a heavy metal, proof of which i s contained i n the fact that the oxidation process was completely inhibited by cyanide.  Further, i t was shown that an e s t e r i f i c a t i o n of phosphoric acid  with organic material i s essential f o r the formation of glucuronic acid, because i t s production i s sensitive to iodoaoetate and to fluoride. Hemingway, Pryde and Williams (?) have studied the effect of cyanide upon surviving tissue.  Conjugation, they state, may depend upon  the c e l l u l a r i n t e g r i t y of the l i v e r or upon the existence enzyme.  of a pre-formed  Potassium cyanide, added to a r t i f i c i a l l y perfused l i v e r outside of  the body prevente d the conjugation of phenol and glucuronic acid thus lending weight to the f i r s t suggestion.  This, however, does not remove the possi-  b i l i t y of an enzyme system taking part i n the conjugation process.  Cyanide  may i n h i b i t such an enzyme system i f one i s presents The suggestion of Hemingway et a l of the part played by an enzyme system i s interesting i n the l i g h t of the recent discovery by Fishman (8) of the enzyme glucuronidase, which he has shown to be s p e c i f i c f o r the hydrolysis of conjugated glucuronides  6  ;  (iv) Site of Formation,  Llpschitz and Bueding (6) nave shown that conjugation takes place p r a c t i c a l l y exclusively i n the l i v e r .  Some conjugation may take place i n  the kidney, (v) Glucuronic acid and the sex Hormones. Several workers have shewn that certain sex hormones are excreted as conjugated glucuronides. Among these are Cohen and Marrian (9) and Yenning and Browne (10).  The former have shown that o e s t r i o l i s linked to glu-  curonic acid by a glucosidic linkage through the aldehyde group of the latter, (vi) Possible r e l a t i o n of glucuronic acid to the cancer problem. The discovery • of the carcinogenic action of the sex hormones and their relationship, structurally, to known chemical compounds has been the i n spiration for a wide variety of work upon these substances.  I t i s now  firmly established that the estrogenic hormones are growth-stimulating chemicals and that the female genital tissues are capable of responding to these chemicals during the greater part of the l i f e of the individual. Work seems to indicate, too, that estrogenic hormones are at least i n d i r e c t l y responsible for mammary tumors.  Their carcinogenic effect i s d i r e c t l y  proportional to t h e i r various physiological a c t i v i t i e s .  Loeb (11) has  presented pertinent evidence to show that some important relationship exists between the sex hormones and mammary cancer i n the female body.  He developed  strains of female mice i n 99% of which appeared spontaneous mammary cancer. Extirpation of the ovaries at a s u f f i c i e n t l y early period reduced the cancer rate to zero.  Lacassagne  (12) produced mammary cancer i n male mice,which  would not normally have developed i t , b y weekly injections of crystalline female sex hormone preparations. Burrows (13) observed that mice injected  with oestrone developed cystic matsophathy, a condition regarded as a preparatory stage toward cancer.  Other ©estrogenic substances produced  similar results. It has been shown many times (14) that various types of polycyclic hydrocarbons have carcinogenic properties.  Whatever t h e i r direct or indirect  role may be i n the formation of malignant growths, i t i s clear that they have two characteristics i n common.  F i r s t they convert normal c e l l s into new  types, by an as yet unknown process. place • i n the animal organism.  These new types are without a natural  And secondly they are insoluble i n body f l u i d s .  Studies have been made on such carcinogenic hydrocarbons as, 1,2,5,6, dibenzanthracene and anthracene i t s e l f with a view to determining what mechanism, i f any exists i n normal tissue to affect t h e i r detoxification. Boyland and Levi (15) have shown that normal tissues convert anthracene into at least three different compounds. ,o-Ct,Ht  Ob  H> OH  HC-NH(cHscq) t iCooH  No proof i s offered as to whether these compounds represent successive steps i n the metabolism of anthracene or whether they are formed independently of each other.  Regarding the f i r s t of these mechanisms, the development of the  glucuronide 1,2 dihydroxy 1,2 dihydroanthracene glucuronide, we have a reaction which has an analogy i n the metabolism of sex hormones; that i s the reaction of two adjacent secondary hydroxyl groups with glucurone to form a glucoside.  linkage rendering*a glucuronide with water soluble properties.  Oestriol  i s so converted into o e s t r i o l glucuronide c f ( 3 /  O- C<i W<?d&  and excreted as such from the animal body. Many substances normally produced i n the body are carcinogenic or are readily converted into carcinogenic substances. 5-methyl 1,2 benzanthracene  Methyl cholanthrene, a  containing a 5 carbon r i n g attached to the s i x  and ten position was found to be very potent and i s s i g n i f i c a n t i n view of i t s resemblance  to the b i l e acids.  Wieland and Dane (16) demonstrated  that  b i l e acids can be transformed into methyl cholanthrene by chemical means. It i s interesting to note, too, that methyl cholanthrene resembles the sterols and the sex hormones i n i t s f i v e membered ring structure and i t i s conceivable that methyl cholanthrene may be a degradation product of these substances. Acetyl choline, a: substance set free by the so-called cholinergic nerves of the sympathetic nervous system has been found highly carcinogenic. I f administered to animals subcutaneously i t produces osteo sarcoma. Work has been done to determine the relationship between the action of oestrone and the female sex hormones generally, and the direct carcinogenic  action of hydro.carbons. i t s action due  Does ©estrone act by direct carcinogenesis or i s  to i t s physiological function under abnormal conditions?  It i s claimed that the mechanism of the production of carcinoma by carcinogenic hydrocarbons and by oestrone i s entirely d i f f e r e n t . I t has been shown that oestrone does not act through i t s polycyclio structure.  Stilbestrol  s  an oestrogenic substance of greater power than oestrone has been prepared by Doads, Goldberg, Lawson and Robinson (17).  This substance acts l i k e  oestrone, i s highly carcinogenic and yet lacks the polycyclic structure. Does this necessarily show that i t i s the p o l y c y c l i c structure of hydrocarbons or of sex hormones which causes carcinogenicity?  And does i t show  that the sex hormones are d i f f e r e n t i n t h e i r action to that of the hydrocarbons?  It i s suggested t h a t . i t does not, because so many substances of  d i s s i m i l a r structure have high carcinogenic a c t i v i t y .  Amino, azo and n i t r o  compounds have been shown to be carcinogenic by Waters (18) and Shear (19). When so many diverse substances, organic and inorganic, have been shown to be carcinogenic, i t would seem that chemical structure i s not primarily associated with carcinogenesis. A mechanism of carcinogenesis has been suggested.  The work of Warburg  (20) has shown that malignant tissue d i f f e r s from', most normal tissues i n producing more l a c t i c acid than i t i s able to oxidize.  K e i l i n (21) has  shown that the respiratory system of most c e l l s can be divided into three parts: 1) Indophenol oxidase, which activates oxygen. 2) Cytochrome, a r e v e r s i b l y reducible pigment which acts as an oxygen carriers 3) The dehydrogenases, e.g. l a c t i c acid dehydrogenase which activates l a c t i c acid so that i t can reduce cytochrome or methylene blue.  The respiration of tumors i s greatly increased by the addition of pphenylene diamine showing that there i s ample indophenol oxidase or "respiratory enzyme". examined.  Cytochrome has been found i n most malignant tissues  Therefore the defect of malignant tissue may be a deficiency i n  dehydrogenases.  Carcinogenic hydrocarbons have been shown to i n h i b i t the  dehydrogenases, which, when absent allow the l o c a l accumulation of l a c t i c acid.  Further, i t i s known that tumor c e l l s survive and grow i n media con-  taining l a c t i c acid, whereas normal c e l l s may be inhibited by the presence of. this substance.  Tumor tissues have a mixed metabolism and produce l a c t i c  acid both i n the presence and absence of oxygen,  Normal tissues produce  l i t t l e or no l a c t i c acid aerobically and must have free oxygen to l i v e . I f c e l l s i n a certain tissue should become malignant due to say the presence of excess sex hormones, they would be able t o grow and reproduce to the disadvantage of surrounding normal c e l l s , _-, Whether chemical carcinogens, including the sex hormones, act as such  9  or whether they undergo chemical changes i n the tissue before they acquire carcinogenic properties i s not known but there should be some mechanism i n the body which removes toxic substances and excess sex hormones before they become carcinogenic. At any rate, the body contains i n i t s tissues definite growth activators and i n h i b i t o r s .  Murphy and Sturm (22), (23) have isolated  from placenta and embryonic skin, substances which d e f i n i t e l y inhibited transplanted and tumorous growth.  The mammary gland i n the pre-lactating  stage yielded a f r a c t i o n which gave a marked stimulation to tumor growth. It i s possible that glucuronic acid may have an important role here.  In  the case of o e s t r i o l , a glucurone-oxidase enzyme system has been observed by Fishman (8), (24) which i s responsible f o r the conjugation of o e s t r i o l with glucurone forming a water-soluble complex, and thus making possible  i t s elimination from the body.  The same or a similar system may exist  ' affecting the hydrolysis of the glucuronide.  I f , for some reason, tissue  c e l l s can no longer conjugate the insoluble hormones to their water-soluble form, or i f the enzyme balance i s upset so that hydrolysis of the glucuronides occurs at a greater rate than t h e i r synthesis, then a preponderance  of i n -  soluble carcinogenic compounds w i l l result which may have their effect upon the respiratory system of the c e l l , as suggested above*  This suggestion i s  put forward only with reference to tumors which may be caused by an overaccumulation of female sex hormones i n the animal body. Much evidence has been collected to show that carcinogenic substances may be anti-carcinogenic under certain circumstances.  Haddow and Robinson  (25) found that carcinogenic hydrocarbons inhibited the growth of Jensen and Walker tumors.  N i t t a (£6) used sex hormones to i n h i b i t tumor growths.  I f , asswas suggested before, the enzyme system balance has been upset, so that hydrolysis of conjugated glucuronides occurs at a greater rate than, t h e i r synthesis, then carcinogenic agents may act anti-carcinogenically by restoring the enzyme balance. Work on t h i s phase of the problem has not been attempted, but several approaches  to a solution might be applied.  1) Studies might be made on the enzyme system with regard to i t s a b i l i t y to synthesize or hydrolyze glucuronides and on the effect on the r e v e r s i b i l i t y of the reaction of various activators and i n h i b i t o r s . 2) By the method of tissue culture to t r y to produce malignant c e l l s i n v i t r o by means of carcinogenic agents including sex hormones and to study the effect of glucuronic acid and the enzyme glucuronidase upon the production of such a system* 3) Investigate the production of cancer carcinogenically on the l i v e  - 11 animal i n two c a s e s — f i r s t where glucuronic acid i s administered along with the carcinogen and second where the carcinogenic agent i s applied alone* The effect of glucuronic acid might be t r i e d also upon a s t r a i n of mice with a knoan high incidence of cancer. 4) Study the effect of activators and inhibitors on the animals capacity to produce the enzyme glucuronidase. I t i s not to be concluded from the above remarks that i t i s believed that glucuronic acid w i l l solve the cancer problem. are many and involved,  The factors concerned  For example, l i t t l e i s known concerning the role of  the p i t u i t a r y gland and i t s secretions, but they must have an important function, controlling as they do the whole endocrine system of the body. It seems possible, however, that glucuronic acid acting i n a detoxification role may be a l i n k i n the chain of events.  I  •"  - •• - 12 'II.  HSvTBW Off METHODS Off SYNTHESIS.  (1) Biosynthesis Quick (27) has been a pioneer worker i n the biochemical production of glucuronic acid.  He condemns menthol as a glucuronogenic drug when fed to  rabbits due to d i f f i c u l t y i n administering the substance and the labor i n volved i n obtaining large quantities of glucuronic acid. destroy menthol or conjugated menthol.  Dogs are able to  The method f i n a l l y evolved by Quick  was to feed five grams of borneol daily to each of several dogs; to isolate the conjugated glucuronide from the urine as the zinc salt and then to prepare the free acid from this s a l t .  By this method, Quick was able to obtain  one gram of zinc s a l t f o r every gram of borneol fed and a f i n a l y i e l d of glucuronic acid of over 80$ of the theoretical. In 1911, Bang (23) prepared menthol glucuronic acid from rabbits. I t consisted i n feeding rabbits two grams of menthol by stomach tube, collecting the"- urine f o r twenty-four hours and extracting the ammonium s a l t by h a l f saturation with ammonium sulphate. Recently, this method has been modified by Williams (29) so that he obtained a y i e l d of 1.4 grams of the crude ammonium menthol glucuronate for every gram of dl-menthol fed.  Williams (30) investigated the use of other  glucuronogenic drugs and gives the following relative values of materials for conjugation with glucuronic acid i n the rabbit. 1-menthol dl-menthol d-menthol  48$ conjugation 59$ conjugation 70$ conjugation  Concerning the r e l a t i v e merits of the preparations of glucuronic acid from dogs and from rabbits as presented by Quick and Williams, respectively, the following may be said.  1.  The method using dogs has the advantage of ease of administration  of the glucuronogenic drug. 2.  Larger quantities can be produced by using dogs as they can receive  borneol every day with no apparent 3.  ill-effects.  Quick,using dogs and borneol reports a better y i e l d than does  Williams using rabbits and menthol. 4.  Quick's method has the advantage of ease of separation of the zinc  s a l t from the urine.  The separation i s more complete than i n Williams  9  separation using ammonium sulphate• 5.  Rabbits are cheaper to buy, more cheaply fed and easier to look  after than dogs.  Larger numbers of them can be kept,  (ii) a)  Chemical methods of synthesis,  oxidation of Glucose. Nef (31) reported the following products as a result of the oxidation  of-d-glucose i n the presence of copper hydroxide i n alkaline solution. 114 gr. d-glucose gave 3.83  gr. CO*,  14.71  gr. glyceric acid, 30 gr. t r i -  oxybutyric acid and 30 gr. of hexonic acids. Acids formed varied with the concentration of alkali? present. K i l l i a n i (32) oxidized glucose (100 gr.) with n i t r i c acid (.8 vold-1.2) for fourteen days and obtained 10.1 gr, "of what i s apparently calcium glucuronate".  K i l l i a n i makes two important comments:  l ) only 1.333  atoms  of oxygen per mole of glucose was used instead of the two atoms calculated for the reaction; hence the regeneration of n i t r i c acid which took place must play a material part i n the reaction. 2) Glucuronic acid i t s e l f i s very stable towards n i t r i c acid (d = 1.2) at room temperature,  f o r the calcium s a l t  precipitated from water solution contained only a minimal amount of calcium saccharate«  J o l l e s (33) oxidized dextrose 37°for 144 hours.  (2$ solution) with H 0 (12 v o l . %) at a  a  The unused glucose was removed with yeast.  Glucuronic  acid was removed as the lead s a l t , the y i e l d being described as poor, b) Oxidation of Gluoosides. Glucose and glucosides were suggested as possible primary compounds for the preparation of glucuronic acid by Fischer and P i l o t y (34).  Fischer  suggested that one source of glucuronic acid i n the body might be through the oxidation of glucosides.  Toxic substances introduced into or formed i n  the body united with glucose to form glucosides. produced glucuronides. that this i s not the  Subsequent oxidation  More recent work, as indicated above seems to show  case.  Smolensk! (35) attempted to apply Fischer's idea to a method for the chemical synthesis of glucuronic acid.  °e Methyl glu.coside oxidized by means  of bromine and sodium carbonate or by hydrogen peroxide i n the presence of f e r r i c hydroxide produced  methyl glucuronide i n yields up to 30$.  Isolation  was by means of the brucine s a l t . Jackson and Hudson (36) studied the cleavage of the carbon chain of glucosides by oxidation.  The oxidation of <methyl d-glucoside with barium  hypobromite produced a dibasic acid which was isolated as i t s crystalline strontium s a l t .  Yields of 65-70$ were obtained.  Periodic acid as an  oxidizing agent produced similar results. The oxidation of maltose as a natural glucoside by G l a t t f i e l d and Hanke (37) gives further evidence of the destruction of the glucose molecule on oxidation.  Using hydrogen peroxide as an oxidizing agent, a great variety  of substances was obtained including formic acid, glucosido acids, g l y c o l l i c acid and oxalic acid.  - 15 Craik (33) studied the mechanism of the oxidation of t y p i c a l carbohydrates with hydrogen peroxide.  Maltose was not attacked by hydrogen per-  oxide except i n the presence of ferrous sulphate.  When oxidation did occur  maltose was attacked f i r s t at the reducing group giving a maltobionic acid which, being f a i r l y strong, caused hydrolysis of the unchanged sugar. Subsequently, oxidation of the fragments occurred giving a great variety of products, Bergmann and Wolff (39) oxidized <menthol glucoside with bromine i n the presence of pyridine and normal sodium hydroxide. «c menthol glucuronide was  A y i e l d of 7.5$ of  obtained.  c) Reduction of the lactone of saccharic acid. Fischer and P i l o t y (34).obtained glucuronic acid i n yields up to 10$ by reducing the lactone of saccharic acid with sodium- amalgam. d) E l e c t r o l y t i c method. Leutgoeb and Heinrich (40) employed an e l e c t r o l y t i c method for the oxidation of «c methyl glucoside. peroxide.  The ultimate oxidizing agent was hydrogen  No catalyst was employed.  cinchonine s a l t .  Extraction was by means of the  Eleven atmospheres pressure was employed.  The y i e l d  obtained was i n the neighbourhood of 20.2$. e) Pure chemical synthesis. Stacey (41) devised a method f o r the oxidation of the sixth carbon; atom of glucose a f t e r protecting the remaining carbon atoms with acetyl groups.  Glucose was converted into 6 t r i t y l penta-aoetyl glucose».  The t r i t y l group was then removed and the resulting penta-acetyl glucose oxidized at the six-th carbon atom w i t h potassium permanganate.  The y i e l d  obtained was 20$ over a l l . Zervos and Sessler (42) started with acetone glucose and converted i t  to 1,2 acetone "3,5 benzylidine «c , d-glucofuranose.  Oxidation by cold  alkaline potassium permanganate produced acetone-benzylideneglucuronic  acid  which when heated t o 100°for one hour i n 50$ alcoholic hydrochloric acid solution produced d-glucurone.  Five grams of acetone glucose produced .9  gr. glucurone* Consideration and comparison of the above methods indicate that none i s highly satisfactory.  Yields are small i n a l l cases.  The last two  mentioned, those of Stacey and Zervos and Sessler, are beautiful examples of organic synthesis, but much too involved to be considered as p r a c t i c a l methods f o r the commercial preparation of the substance.  The method i n -  volving the e l e c t r o l y s i s of methyl glucoside requires expensive apparatus, while the y i e l d i s r e l a t i v e l y low. of  9  Smolensk! s method involving the oxidation  methyl glucoside with hydrogen peroxide plus f e r r i c hydroxide with the  production of a 30$ y i e l d of glucuronic acid i s interesting.  Unfortunately  only the abstract of the paper i s available and none of the details of the method employed are known.  -17 III.  PROPERTIES OE GLUCURONIC ACID AND ITS COMPOUNDS COMPOUND  OPTICAL ROTATION  MELTING POINT  1-glucurone  REFERENCE  c  169-172  d-glucurone  34 43  [<<f ~fl8.55 D  fafter 3 min.  (<£Wl6.05 in H 0 a  d-glucuronic acid after 3 hrs.  W r - +36 i n H*0  zn. dl-borneol glucuronate .2 H^O  Deeomp. 206°  1-borneol glucuronic acid (anhyd.) fc^- -69.03 i n Hg.0 d-borneol glucuronic acid (anhyd.) [<0'°- -37.02  162-3~  ^ - t r i a c e t y l glucurone  194-195  44  110-112°  44  0  164-5  0  + 84.1 i n CHC1 (k.]^-203.6 i n CHC1  3  rf-triacetyl  glucurone  3 5  diacetyl chloro-glucurone  107.5-108.5  45  130-131'  45  i n CHGI3 +•1.60  'after 2 min. Diacetylglucurone  +• 0.85  after 25 min. S i l v e r glucuronate  W -t.19.2  46  ^+44.6  46  0  i n HjO Methyl glucuronate 3tetraacetyl glucuronic acid methyl ester.  +8. 7 i n CHCI3  ettetraacetyl glucuronic acid methyl ester.  ll^]o-+98.0 i n CHC1  46  178  111-112  46  150.5-151,5°  46  c  3  1-chloro-triacetyl glucuronic acid .^=-16.7 i n CHCI3 methyl ester. C ~ . &fo Diacetyl methyl glucoside of glucurone.  D4,=-'-ll25 i n CHC1 : C .6$  110-111'  47  T r i a c e t y l methyl glucoside of glucuronic acid methyl ester.  [«<f-+54.0 i n CHCI3 C=.6$  118  47  0  - 18 COMPOUND  OPTICAL ROTATION  Diacetyl p-nitrobenzyl glucoside of Glucurone.  MELTING POINT  .REFERENCE  133-134°  47  175-176  47  i n CHC1 0 -1%  3  T r i a c e t y l p-nitrobenzl glucoside of glucuronic acid methyl ester.  M?"—57.8 i n CHCI3  C = .6$ «<• T r i a c e t y l chloroglucuronic acid methyl ester.  Mo-+168.7  99-100'  48  104-105'  48  149-150'  48  167-168'  48  112.5-114'  49  116-119  49  0  49  i n CHCI3  0 = .5$ 06 t r i a c e t y l bromoglucuronic acid methyl ester.  t^^+198.0  £ Methyl glucoside of T r i a c e t y l glucuronic acid Methyl ester.  C4> = -28.9  i n CHCI3 0 = .556  i n CHCI3  C = .7$ & p-Nitro benzyl glucoside of Glucuronic acid methyl ester.  t O ^ + 63.2 i n Hz.0 C =1$  1,2,3,4 Diacetone galactose 6 2,3,4 Tr-iacetyl glucuronide Methyl Ester.  rf--63.0 i n CHGI3  C =2.0$ Initial Galactose 6, /3 -Glucuronide Final-2Hrs. ,C =lfo  i n H«0  f I n i t i a l - 6 Min.... s^ll9 L«d"--2.90 Methyl Ester of Galactose 6/3 Glucuronide.  Final C =4.0 % i n H^O  Heptaacetyl Methyl Ester of Galactose 6/3 Glucuronide.  202-203'  49  131-132'  50  i n CHGI3  0 =3$ Trimethyl glucurone.  M-v-197.5 " 'b - .76 i n H*P. f  « ;  , IT. . EXPERIMENTAL WORE,  a) Biosynthesis. Preparatory to attempting a chemical synthesis, i t was necessary to obtain a pure sample of glucuronic acid or of one of i t s compounds.  For  this purpose the biosynthesis of Williams (29) was followed with certain modifications. D i f f i c u l t i e s encountered i n t h i s method and the modifications applied are as follows. Rabbits (female) weighing between S§- and 3 kg. were used. They were housed i n a cage having a wire top and bottom.  The cage was  placed i n a wooden stand having a galvanized iron funnel-shaped bottom, of the size of the bottom of the cage. made i n a large erlenneyer.  Urine collections were therefore e a s i l y  A fine copper screen wire was placed between  the cage bottom and the funnel to prevent food or faeces from entering the urine. As neither d or dl-menthol was available, 1-menthol was used throughout a l l experiments.  Previous workers have recommended feeding one gram  of glucuronogenic drug per kg. of body weight. • I t was found that more than this could be given i n the case of 1-menthol. received 4 gr. of 1-menthol at each feeding.  Rabbits weighing 2700 Gr, Feedings i n our case took  place once a week but could be increased to once every three days, To administer 1-menthol to a rabbit i t i s necessary to anaesthetize the animal and then to use an enema tube t o place the compound i n the stomach. The animal was best held by placing i t i n a large round t i n about 18 inches high and 8 inches i n diameter, padded on the inside. by an aspirator. free of peroxides.  Ether was  administered  Care should be taken to use a good grade of ether and one The ether was tested f o r peroxides by acidifying with  acetic acid, adding potassium iodide and starch and heating.  I f present,  peroxides free -the iodine and the starch solution i s colored blue.  If  peroxides are present, they can be removed by d i s t i l l i n g the ether over water and copper wire into a brown bottle containing a copper c o i l , the bottle being kept f u l l to exclude the presence of a i r . The rabbit's mouth was held open by a bone spatula through which a hole had been bored to permit the entrance of the enema tube.  I f the rabbit i s held upright, there  i s l i t t l e p o s s i b i l i t y of forcing the tube down the trachea even when the rabbit i s under ether. The rabbit should be starved for 24 hours before being given the menthol. Following Williams' suggestion the menthol was made into an emulsion with about 30 c.c. of warm water.  D i f f i c u l t y was experienced at t h i s point.  The rabbits came out of the ether w i t h i n five minutes of the administration of the menthol.  Within one h a l f hour a f t e r the rabbits came out of the  ether, they lapsed into a state of coma, which lasted for about three hours in.'-.the case of one rabbit and two hours i n the case of the others.  The  animals appeared to be groggy and sleepy.  A l l voluntary action was  inhibited  and reflex action of the eye was  The rabbits appeared t o be ex-  stopped.  tremely hungry especially when coming out of the coma. suffered, as f a r as could be determined by observation.  No i l l effects were This phenomenon  happened at each feeding and was thought at f i r s t to be due to the ether used, but this p o s s i b i l i t y was removed by freshly d i s t i l l i n g  the ether  and thus removing peroxides. An attempt was made to discover the cause of the phenomenon just described.  I t was  thought that i t might be due to shock caused by removal  of glucose from the blood stream of the animal.  I t was thought too that  information on t h i s point would indicate whether or not body glucose i s used by the animal i n synthesizing glucuronic acid„  The following experiment was carried out using three rabbits. Rabbit No. I. 1G c. c. of blood were withdrawn from the marginal vein. 1-menthol were fed by the method described above.  Four grams of  I t was noted that this  animal did not go into a state of coma as soon as those from which no blood had been withdrawn, but after one and one half hours i t lapsed into a condition resembling death. decreased..  Even the heart was  slowed down and body heat  At this point an additional 5 c.c. of blood'were withdrawn from  the marginal vein.  Later, when the rabbit had been i n the state of coma  for about, two hours, i t was given 3 gr. of glucose i n water by stomach tube. This was apparently without e f f e c t , as the animal did not completely recover f o r another four hours. Analyses f o r blood sugar were made on the two samples, by the method •of Somogyi-Shaffer-Hartmann the two samples.  (51).  No significant difference was  found i n  I t was concluded, therefore, that the coma was not caused  by the shock of depleting the blood of glucose.  Menthol apparently does not  unite with glucose f i r s t to form a glucoside as was suggested by Fischer  e  Rabbit No. 2 4 gr. of 1-menthol and 4.6 gr. of glucose i n a water emulsion were administered t o a rabbit weighing 2800 gr.  This animal did not go into a  coma as rapidly and recovered more quickly than i n previous times when no glucose was given.  This was not interpreted as supplying evidence that  body glucose went to the formation of glucuronic acid. Rabbit No. 3. The effect of borneol on rabbits was t r i e d . i t vjould be advantageous, soluble zinc s a l t .  I f borneol could be used  because of the ease of separating out the i n -  Accordingly, 3 gr. of 1-borneol dissolved i n olive o i l  - 22 was fed to one rabbit.  The effect was different than i n the case of menthol.  The animal did not go into a state of coma, but suffered i n t e s t i n a l spasms which must have been p a i n f u l .  The effect produced by borneol lasted much  longer than that produced by menthol three days being required f o r complete recovery.  During this time, the rabbit suffered a loss of appetite and  l o s t much weight.  No conjugated glucuronide was recovered from the urine.  I t was decided that either borneol i s metabolized or that i t passes through the rabbit unchanged, and that borneol i s not a satisfactory glucuronogenic drug f o r rabbits. An attempt was made to prevent the oncoming of the state of coma after administration of menthol.  I t was found that 4 gr. of 1-menthol could be  administered to a rabbit of 2.5-3.0 kg. without producing the after effects of the coma, i f the menthol was dissolved i n olive o i l .  Slight dizziness  .did occur i n some cases when olive o i l was used, but nothing l i k e that produced by menthol alone i n water.  A l l l a t t e r experiments were cafried  out, using olive o i l as a solvent f o r menthol.  The only explanation for the  effect of the olive o i l i s one of slower absorption.  The urine should be  collected over a longer period of time when olive o i l i s used. The method of treatment of urine was the same as given by Williams (29). Yields of crude ammonium menthol glucuronate are reported by the l a t t e r as being 1.4 gr. of crude glucuronate f o r every gram of d-1 menthol fed, which conjugates to the extent of 59$. Over twelve 1-menthol administrations yielded one gram of crude ammonium menthol glucuronate per gram of 1-menthol fed, which corresponds to a 43$ conjugation of 1-menthol as reported by Williams.  Yields were not affected by using olive o i l i n the administration  of the menthol*  Living/and Post-mortem examination of the rabbits. At  the end of a five month period which included f i f t e e n administrations  of lr-menthol, the rabbits were i n excellent health and appeared to have suffered no i l l - e f f e c t s . Post-mortem and h i s t o l o g i c a l examination was carried out. Parts examined viere oesophagus, stomach, small and large intestine, l i v e r , lungs and kidneys. the  lungs.  fed menthol.  Gross examination showed one rabbit to have a haemorrhage of  This was probably not primarily due to the animal having been The l i v e r showed one white area on the edge of one lobe.  This i s the f i r s t sign of l i v e r disintegration.  The inside wall of the  stomach was very carefully examined for the presence of stomach ulcers.  One  area which very much resembled the so-called chronic or round ulcer was found, about the middle of the stomach. Conclusions concerning the Biosynthesis. Exact methods of procedure f o r the production of glucuronic acid from rabbits has been outlined.  The biochemical method using either dogs or  rabbits offers probably the best means now available of synthesizing glucuronic acid. I t . i s produced r e l a t i v e l y cheaply and i n good y i e l d , and i s separated from the urine as a stable compound. The process of removing the glucuronic acid from the secreted compound offers no d i f f i c u l t i e s and can be carried through p r a c t i c a l l y quantitatively. The glucuronogenie drug i s recoverable and can be used over again. b) Chemical synthesis. In t h i s investigation i t was proposed to prepare glucuronic acid from glucose by oxidation after f i r s t protecting the aldehyde group by the formation of some condensation product, such as the phenyl hydrazone,  - 24 oxime or glucoside,  The primary alcohol group would then be oxidized to a  carboxyl group and the resulting compound hydrolyzed to give the aldehyde group back again, thereby forming glucuronic acid. The. glucose phenylhydrazone was made according to the method of Jacob! (52).  Glucose 2,4 d i n i t r o phenylhydrasone was also prepared.  Neither  hydrazone was found to be satisfactory f o r oxidation to glucuronic acid due  to their i n s t a b i l i t y . Glucose oxime was prepared according to Wohl (53) by Gwyn (54). cC Methyl glucoside was prepared by the method of Patterson and Robertson  (55).  This compound has been shown to be very suitable for the purpose,  1  being stable i n so f a r as the linkage between the methyl alcohol and the glucose molecule i s concerned. The natural glucoside maltose was given consideration as a possible starting point. Borneol glucoside was thought t o provide a good starting point,as by keeping zinc ion i n the oxidation mixture i t would be possible to precipitate out zinc borneol glucuronate as soon as any borneol glucuronide was formed. The compound borneol glucoside was prepared accordingly.  The tetra-acetyl  brom derivative of glucose was f i r s t prepared according to the method of Koenigs and Knorr (56).  This product was then condensed with borneol i n  the presence of s i l v e r carbonate by the method of Fischer and Raske (57). The following modifications to the method of synthesizing borneol glucoside were made.  The procedure for making tetraacetyl brom glucose  requires at least ten hours f o r completion according to the method above. I f ether i s added to the reaction to prevent the contents from becoming too viscous a mercury seal s t i r r e r can be used to keep the reactants i n motion.  - 25 Thus, place glucose, glass beads, (small) and acetyl bromide i n a claisen flask with the sidearm removed.  Add sufficient ether to prevent the re-  action mixture from becoming too viscous.  Into the straight arm of the  claisen flask insert a mercury seal s t i r r e r and into the other a calcium chloride tube to permit escape of hydrogen bromide and prevent the entrance of water vapour. of l i g h t .  The flask i s then-completely  covered to prevent entrance  The temperature should not go above 5°C,  I f the reaction becomes  too vigorous as indicated by the evolution of hydrogen bromide i t can be slowed down by lowering the temperature of the mixture.  S t i r r i n g i s con-  tinued u n t i l a l l the glucose has gone into solution which should be the case i n about h a l f an hour.  The y i e l d i n this step i s about 60$ and i n  the second step about 50$, so that the overall y i e l d i s only 30$.  Difficulty  was experienced i n oxidizing the compound, p a r t l y due to i t s sparing solub i l i t y i n water.  In view of this and the d i f f i c u l t y involved i n i t s  preparation, coupled with a low y i e l d , i t was decided not to proceed further i n the study of borneol glucoside as a possible oxidizable substance. The investigation was carried out along theoretical lines i n an endeavour t o discover the value of different oxidizable substances  and  different oxidizing agents and to discover the effect of pressure, temperature and various catalysts.  Experiments on these phases of the problem  involved the U3e of Tollen's naphthoresorcinol test as modified by Maughan, Evelyn and Brown (53) and more recently by Mozolowski (59) and were carried out on a small scale.  When the problem of separation was attempted,  d i f f i c u l t i e s were encountered.  I f glucuronic acid i s present even i n  f a i r l y complex mixtures, i t should be r e l a t i v e l y simple to separate. Therefore, i n a b i l i t y to separate yields indicated by maphthoresorcinol  led to a study of the s p e c i f i c i t y of the reagent. The Klett-Summerson photo-electric colorimeter was standardized as follows.  Solutions of p u r i f i e d ammonium menthol glucuronate(M.P. 135°  with decomp.) were made up so that they contained the equivalent of .01, .02, and .05 mg. of glucuronic acid per 2 c . c of solution.  2 c.c. portions  of these solutions were taken and to them were added 2 c.c, portions of a 0.2$ solution of naphthoresorcinol reagent and 2 c.c. portions of concentrated hydrochloric acid.  The mixtures were heated on a water hath  f o r t h i r t y minutes and then cooled i n i c e water f o r ten minutes.  A blank  test consisting of 2 c.c. of d i s t i l l e d water, 2 c.c. of cone, hydrochloric acid and 2 c.c. reagent should be run at the same time.  After cooling for  ten minutes the coloring matter was extracted with three 5 c,c. portions of ether containing about 1 5 $ absolute alcohol. the mark i n calibrated ICLett colorimeter  Volumes were made up to  tunes.  Colorimeter readings obtained with different samples of naphthoresorcinol are as follows. TABLE I. Glucuronic Acid/2 c.c. '  1  .01  ^  32.5  .02 .05  ELett  55.0 -  82.0  .0125  45.0  .025:.  36.4  .05  65.0  .025  45.0  .04 .05  58.0 68.0  3.  It i s seen, therefore, that supposedly i d e n t i c a l solutions of naphthoresorcinol produced different colorimetric readings, although the three curves produced are p a r a l l e l l i n e s .  It was necessary to recalibrate the  instrument f o r each new solution of reagent. (i) Oxidizing Agents. Several oxidizing agents were t r i e d .  Hydrogen peroxide was t r i e d f i r s t  because of the ease with which any excess can be removed and because of i t s gentle oxidizing power.  Any excess hydrogen peroxide was removed from the  oxidation mixture before the naphthoresorcinol test was applied. effect of hydrogen peroxide on the test was determined.  The  Results are as  follows* TABLE I I . Solution Tested  Result  1.  Dilute H ^  solution  .green color extracted i n ether layer.-  2.  Solution of glucuronic acid  reddish purple i n ether layer.  3.  10 drops 30$ H^O^plus 2 c.c. of (2) above  d i r t y green color i n ether layer.  I t was concluded therefore that hydrogen peroxide destroys the naphthoresorcinol test. The presence or absence of hydrogen peroxide i n reaction mixtures was determined by the following test.  1 c.c. of the solution to be tested  was added to 1 e.c. of dilute potassium aidhromate solution and to the mixture was added 2 or 3 drops of one normal sulphuric acid. was immediately extracted with ether.  The solution  A blue color i n the ether .layer  indicated the presence of hydrogen peroxide.  Methods of destroying excess hydrogen peroxide were studied.  Since an  aqueous solution of hydrogen peroxide i s stabilized toward heat by the presence of acids and since oxidation produces acidic substances, b o i l i n g w i l l not remove excess hydrogen peroxide.  D i s t i l l a t i o n of the aqueous  part of an oxidation mixture did not remove a l l of the excess oxidizing agent even when the solution was taken down to a syrup.  Hydrogen peroxide  can be destroyed by manganese dioxide i n neutral s o l u t i o n .  The method  followed i n reaction mixtures to decompose hydrogen peroxide was to adjust H  the solution to P 7 with a suitable dilute base and then to add excess manganese dioxide which was l a t e r removed is catalytic,  by f i l t e r i n g .  The action here  A second c a t a l y t i c method of removing hydrogen peroxide  was to add p l a t i n i z e d asbestos to the s o l u t i o n removing the l a t t e r by filtration. Hydrogen peroxide was used t o oxidize methyl glucoside, glucose oxime fBy Gwyn 54) and maltose.  The greatest colorimetric reading was obtained  when hydrogen peroxide was used i n the r a t i o of four of oxidizing agent to one of the oxidizable substance, A t y p i c a l experiment performed i s as follows. glucoside were dissolved i n 80 c.c. of water.  5 grams of<* methyl  To the solution was added  5 c.c. f e r r i c chloride solution (,0001 gr./c.c,)< and 7 c.c. of 30$ hydrogen peroxide.  The mixture was placed i n a pressure flask, sealed, and main-  tained at a temperature of 50°C f o r ninety-five hours,  A test by the  photo-electric colorimeter, using naphthoresorcinol, showed a 10.78$ conversion to glucuronic acid.  10 c.c. of the mixture was t i t r a t e d with  .1126 N so-diuin hydroxide and found to require 31,6 c.c. showing that the entire reaction mixture contained .0331 equivalents of acid.  Next, 10 c.c.  of the mixture was d i s t i l l e d under vacuum and the d i s t i l l a t e titrated with .1126 N sodium hydroxide.  20.6 c.c. of the base were required showing the  presence of .0214 equivalents of v o l a t i l e acids. therefore equal to .0117 equivalents, side were used i n the reaction.  Non-volatile acid was  .0258 equivalents of oc methyl gluco-  These results indicate that the glucose  molecule i s s p l i t probably at carbon atom three, with the formation of formaldehyde and formic acid. A t y p i c a l experiment employing maltose Is as follows. were dissolved i n 50 c.c. of water.  12 gr, of maltose  To this solution was added 10.2 c.c.  of 30$ hydrogen peroxide and 5 c.c. f e r r i c chloride (.0001 gr./c.c.). The mixture was placed i n a pressure flask, sealed,and kept at 50°G for 30 hours i n a constant temperature oven.  A test with naphthoresorcinol  indicated the presence of .555 gr. of glucuronic acid. Conclusions concerning hydrogen peroxide. 1}  H Oihas a d i s t i n c t advantage i n being cheap.  2)  Any excess i s e a s i l y removed.  3)  I f used at room temperature i t s action i s slow.  2  I f used at elevated  temperatures, other reactions are aided as well as the oxidation of the primary alcohol group. 4)  Apparently there are several competing reactions, namely* a) dxidation of the 6th carbon atom. b) Breakdown of the glucose molecule at the third carbon atom, °) Possible hydrolysis of <*> methyl glucoside by acids formed by oxidation with hydrogen peroxide,, Use of barium iodide and iodine as an oxidizing agent. G-oebel (60) oxidized glucose with iodine i n an alkaline solution and  obtained a 91 % y i e l d of gluconic acid.  I t was thought that the same method  might be applied to the oxidation of < methyl glucoside to obtain methyl glucuronide.  The procedure was as follows.  were dissolved i n water.  4.5 gr. of <A methyl glucoside  To t h i s solution was added 25 gr. of barium  iodide (Merck) and 25.4 gr. iodine and the whole made up to 350 c.c. The mixture was warmed to 50°C and 500 c.c. of .4 1 barium hydroxide was added from a dropping funnel over a period of f i f t e e n minutes at a constant rate of flow the solution being mechanically s t i r r e d during this operation. The solution was s t i r r e d for f i f t e e n minutes a f t e r the addition of the barium hydroxide and was then a c i d i f i e d with 9.25 c.c, cone, sulphuric acid,  75 gr. basic lead carbonate were added immediately with vigorous  mechanical s t i r r i n g , which was continued u n t i l the solution reached a P  H  of 5 when the precipitate was allowed to s e t t l s , then f i l t e r e d o f f and washed several times with water. with naphthoresorcinol reagent.  The resulting f i l t r a t e gave a good test The f i l t r a t e was evaporated down to a  small volume under vacuo whereby excess iodine was removed, and a yellow precipitate of lead iodide settled out which was f i l t e r e d o f f . Dilute sulphuric acid was added to precipitate the lead which was removed by centrifugation.  Any hydrogen iodide l e f t was removed by s i l v e r sulphate  and excess s i l v e r by hydrogen sulphide. Sulphate ion was removed by barium hydroxide and the excess barium by means of carbon dioxide.  This  ion-free solution at this stage gave a good naphthoresorcinol test.  The  solution was concentrated to a small volume and then to i t was added several volumes of 95$ alcohol which caused a white precipitate to settle out. Iodine was t r i e d as an oxidizing agent i n the b e l i e f that a stronger  and faster oxidizing agent than hydrogen peroxide would act on the sixth carbon atom of glucose before attacking the rest of the molecule.  By  carefully controlling the time of oxidation, and the temperature of the reaction, and removal of oxidizing agent, i t was hoped to stop the reaction at the c r u c i a l moment. Accordingly, the above procedure was modified as follows,  5 grams of  eC methyl glucoside were dissolved i n 50 c.c. of water and to the solution was added 5 c.c. of f e r r i c chloride (.0001 gr./c.c.). 3.2 gr. of f i n e l y pulverized iodine were added and just enough barium iodide (Merck) to i n i t i a t e solution of the iodine.  The beaker was placed on an ice bath'  and 15.4 gr. of barium hydroxide i n 400 c.c. of water added with constant s t i r r i n g over the course of three days.  At the end of this time a small  excess of barium hydroxide was added to decolorize the solution.  A pre-  c i p i t a t e (probably barium iodate)' was f i l t e r e d o f f and to the f i l t r a t e was added excess lead acetate to remove lead iodide which was separated by filtering.  The remainder of the iodine was removed with s i l v e r acetate.  Lead ion was removed with hydrogen sulphide and barium ion with excess sulphuric acid„  At this point the solution was hydrolyzed for three hours,  after which the excess sulphuric acid was removed with lead acetate and the lead ion with hydrogen sulphide. duced a deep red color.  Tests with naphthoresorcinol pro-  The solution was evaporated to a thick syrup  which on warming s l i g h t l y , c r y s t a l l i z e d to a s o l i d mass were taken up i n hot 95$ ethyl alcohol. dissolve and was f i l t e r e d o f f .  0  These crystals  A small white residue did not  This remained unidentified.  on standing, produced large prismatic needles, constant melting point of 55°C was carried out.  The solution,  Hecrystallization to a The compound apparently  was the meso -form of diethyl tartarate.  Although the f i r s t mentioned  compound was not i d e n t i f i e d there i s reason to-suppose that glyoxylic acid constituted the major portion of the remainder of the. oxidation products. This would account f o r the remarkable naphthoresorcinol test obtained at various points i n the treatment of the reaction mixture,  Bergmann and  Wolff (39) oxidised <c methyl glucoside with bromine i n the presence of barium hydroxide and obtained an excellent y i e l d of glyoxylic acid which they describe as giving a beautiful color with naphthoresorcinol reagent. Conclusions from these and other similar experiments were that iodine i s an unsuitable oxidising agent for the production of glucuronic acid, from methyl glucoside. Iodine i n conjunction with potassium iodide and potassium hydroxide was  also used, and while the naphthoresorcinol test  always provided evidence that glucuronic acid was present, none could be separated out by the usual methods of procedure. Oxidation by potassium permanganate. I t was thought that potassium permanganate might provide a good oxidizing medium i n that i t s strength-is,easily standardized, the manganese i s easily removed, and the potassium salt of glucuronic acid i s easily c r y s t a l l i z a b l e . Accordingly, the following experiment was carried out, glucoside were dissolved i n 50 c.c, of water.  5 grams of <* methyl  To this solution was added  .9 c.c. 18 N sulphuric acid and 35 c.c. of potassium permanganate solution (1.62 gr./c.c.) and the mixture allowed to stand at room temperature.  A  precipitate of manganese dioxide settled out after three hours, while after standing f o r two days, this disappeared and l e f t a clear, colorless solution. Tests with naphthoresorcinol and the ELett colorimeter indicated the presence of 2.24 grams of glucuronic acid.  The products of oxidation were not  - 33 -  i d e n t i f i e d , but glucuronic acid, i f present at a l l , was there only i n vary small amounts'and could not be separated out by the usual methods. • ( i i ) The effect of Pressure. . Considering the fact that Leutgoeb- and Heinrich (40) used eleven atmospheres pressure, i n t h e i r e l e c t r o l y t i c oxidation method, we i n v e s t i gated the effect of pressure on the oxidation of «c methyl glucoside by hydrogen peroxide.  A high pressure w i l l control the rate of decomposition  of hydrogen peroxide according to the reaction  •—s- 2  2 H^Oj.  HrO  -t- Or.  •  which allows the use of a smaller excess of hydrogen peroxide than that -  which was- found to be the most'.advantageous under atmospheric pressure. It was thought too, that pressure might prevent the s p l i t t i n g of the glucose molecule which has been s hown to take place by Jackson and Hudson (36). Two oxidation mixtures were prepared containing 5 grams of methyl glucoside, 80 c.c. of water and 5 c.c. of f e r r i c chloride. peroxide were added at the more a f t e r 43 hours. 132 hours.  3 c.c. of 30$ hydrogen  beginning, 2 c.c. more after 17 hours and 2 c.c.  The mixtures were kept at 50°C for a t o t a l period of  One solution had a c a p i l l a r y tube sealed into the stopper to  keep the pressure at atmospheric without allowing evaporation and the other flask was put under pressure. ..using a car pump and a t i r e valve sealed into the stopper.  Results can be summarized as follows, .  K l e t t reading  $ conversion  Mixture under high pressure  47.8  13.10$  Mixture under medium pressure  43.9  10.78$  Mixture under atmospheric pressure  42.8  10.12$  From these results i t was concluded as a result of colorimetrie determination, that i t i s best to oxidize under pressure, although there  i s r e l a t i v e l y l i t t l e difference obtained under low and high pressure—at least at the pressures obtained i n our mixtures.  Moreover, the color of  the oxidation mixture, when kept under pressure, remained lighter, showing less breakdown of the glucose molecule. ( i i i ) The effect of temperature. The effect of temperature has been investigated.  Increase of temperature  would be expected to speed up the various reactions going on simultaneously i n any mixture which was used.  The question was whether or not an increase  of temperature would favor the reaction involving hydrogen peroxide and the primary alcohol group.  I t was noted that higher temperatures caused  the reactions involved to proceed faster, as noted by the decomposition of the hydrogen peroxide. At elevated temperatures, the mixture turned a dark color indicating deep-seated decomposition of the methyl glucoside molecule.  Aqueous solutions of glucuronic acid are colorless*  I t was  "decided that i t i s best to carry out the oxidation reaction at a temperature i n the neighbourhood of  50°C,  when hydrogen peroxide i s used as the oxidizing  agent. (iv) E f f e c t of P* Experiments were carried out to determine P of the oxidation.  H  changes during the course  Five solutions were prepared each containing 1 . 9 4 grams  of oc methyl glucoside, 1 0 c.c. of water, 1 0 c.c. copper acetate solution (.0001  grams/c.c.) and  3.7  c.c. of  30$  hydrogen peroxide.  The P  H  was ad-  justed to the values l i s t e d below with . 1 N sodium hydroxide. Oxidation was carried out at  40°0  for  12  hours i n sealed pressure flasks.  Solution  P  1  H  adjusted to  P " after oxidation .  6.15  2.22  8.6  2.4  3  6.98  2.22  4  9.5  2.22  5  7*3  2.31  2  •  H  • Change of P over a period of oxidation was next investigated.  The  results are summarized below. TABLE IV. Solution 1)  0 Hrs.  10 c.c. Cu(AC) 10 c.c. H i O 1.5 c.c. H^O*,  R  16|- Hrs.  42j Hrs.  95 Hrs.  solution 6.52  6.58  6.72  8.82  8.46  8.15  6.82  4.72  3.82  7,05  3.75  3.28  6.66  5.50  5*12/  .2) '20 d.c. H 0 t  8.18  1.5 c.c. H 0j. t  3)  .97 Gr. «c methyl glucoside 10 c.c. Cu(AC) solution 10 c.c. HiO 1.5 c.c. H^Oa. t  4)  5)  .9 gr.' *• methyl glucoside 10 c.C. .HiO 10 c.c. CuCACJt solution 2 c.c.  £-321  GH3OH  10 c.c. Hj.0 10 c.c. Cu(AG)jlsolution 1.5 c.c. HiO^.  irom the P  H  4.95  changes noted, i t i s evident that oxidation i s accompanied  by the production  of some acid.  I t i s to be noted that glucose and methyl  glueosfcde give nearly i d e n t i c a l results.  This may be due to the fact that  ;  - 36 -  production of acid causes hydrolysis of the methyl glucoside with subsequent oxidation of glucose i n the solution o r i g i n a l l y containing methyl glucoside. Experiments were conducted to determine the relationship between the P  H  and the amount of glucuronic acid produced as indicated by the naphtho-  resorcinol test.  Five solutions containing one gram of < methyl glucoside  and 2 c.c. of 30$ hydrogen peroxide were prepared and mixed with the additional reagents l i s t e d below. The P  ri  before^ oxidation was adjusted  with .15 N sodium hydroxide. TABLE V. H  P before oxidation  Solution  rf  P after oxidation  Color of solution  eolorimetrio reading Dil.of 1/100  1) 20 c.c. H,0  7,2  2.78  dark amber  48.8  2) 10c. c. H;s0 10c.c. Cu(ACK  6.78  2.82  l i g h t amber dark residue  82.1  3) 15 c.c. Hj.0 5 c.c. Cu(AC)i  6,80  2.80  pale yellow white residue  57.3  4) 10 c.c. H 0 10 c.c. Fe(0H)  3  6.80  2.78  clear red  29.0  5) 15 c.c. EUO 10 c.c. Fe(0H)  3  6.85  2.75  d i r t y brown plus residue  24.0  a  From these results i t was concluded that there i s no correlation between the f i n a l P  H  of the solution and the amount of oxidation as shown by the  Tollen's test. The nature of the acid produced was investigated i n one of the above reaction mixtures.  Number (3) was vacuum d i s t i l l e d .  The d i s t i l l a t e had a  P  ri  of 2.98, reduced hot s i l v e r n i t r a t e solution and gave a red ring test  with r e s o r c i n o l . Therefore, at least some of the acid produced i s v o l a t i l e and has reducing properties. had p r a c t i c a l l y the same P  H  I t i s significant to note that the d i s t i l l a t e as the o r i g i n a l oxidation mixture at the end  of the oxidation period. The following conclusions were reached with regard to P oxidation mixtures of  H  changes i n  methyl glucoside and hydrogen peroxide with f e r r i c  chloride as a catalyst. Temperatures employed were i n the neighbourhood of 50°C and pressures were those developed i n sealed pressure flasks. 1)  Irrespective of to what value the P  H  was adjusted at the beginning of  the oxidation period, the f i n a l value always l i e s between 2.2 and 2.8. 2)  Oxidation may r e s u l t i n the production of an acid strong enough to  hydrolyze methyl glucoside into i t s two components.  Subsequent oxidation  results i n the s p l i t t i n g of the glucose molecule to produce acids of two, three or four carbon atoms. 4)  The acid consists of at l e a s t two f r a c t i o n s f i r s t a non-volatile s  f r a c t i o n and secondly a v o l a t i l e f r a c t i o n which has reducing properties* Formic acid i s suggested as the most l i k e l y . acid produced i s v o l a t i l e .  The larger portion of the  For example, i n one experiment the results  were as follows. ..  -.-Methyl glucoside used-.0258 equivalents Total acid produced  - .0331 equivalents  V o l a t i l e f r a c t i o n of acids - .0214 equivalents Non-volatile fraction of acids - .0117 equivalents (v) Catalysts and their e f f e c t s . I t was hoped that some catalyst could be found which would be specific  - 38 for the oxidation of the primary alcohol group; associated with the sixth carbon atom of the methyl glucoside molecule. Various workers have investigated the use of catalysts i n the oxidation of carbohydrates.  Craik (38) found that maltose was not affected by hydrogen  peroxide except i n the presence of ferrous sulphate.  Smolensk! (35)  claimed  to have oxidized oc methyl glucoside using f e r r i c hydroxide as a catalyst. Copper acetate has been used i n the oxidation of carbohydrates.  I t was  decided therefore;, to investigate a number of metallic ions with regard to t h e i r c a t a l y t i c properties i n the presence of hydrogen peroxide. The catalyst as designated below i s based on mole equivalents of metallic ions i n most suitable ion concentration as found by Gwyn (54)• fa  1  7.4 x 10~ moles  2  14,8 X 10" moles  3  29.6 x 10" moles  6  fe  "  2 c.c. of 30$ hydrogen peroxide, .97 grams of «c methyl glucoside and 35 c.c. of water made up the oxidation mixture.  The flasks containing the  mixtures were sealed and l e f t standing at room temperature i n the dark for one month.  Copper, thallium, selenium, and iron were investigated.  Results  are as follows. TABLE VI. Copper  Klett reading  State of Pressure  c - 1  129  maintained  e--i 2  45  no pressure  c -3  62.5  maintained  - 39 TABLE VI(continued) Thallium  K l e t t reading  State of Pressure  T• - 1  23.5  no pressure  T - 2  25.0  no pressure  T - 3  66.0  maintained  Selenium S - 1  maintained  S - 2  0  none  S ~ 3  12*0  none  Iron  •  F - 1 ' F - . 2  F  -  129.0  •  3  maintained  45  none  62.5  maintained  According to these results, iron and copper seem to be about equal i n their catalytic ieffeet.  Subsequent work showed that iron was more  effective than copper and was therefore used throughout most of the work. In the cases of both copper and iron, the smallest amount of metal proved to be the most e f f e c t i v e .  In the case of iron 7,4 x 1 0  _ c  moles was the  most effective concentration. I t i s to be noted,too, that pressure had considerable e f f e c t , the higher pressures giving greater colorimetric readings. tvi)  S p e c i f i c i t y of naphthoresorcinol test.  As stated at the outset, the problem was approached from the theoretical point of view.  A l l conditions affecting the oxidation process were con-  sidered i n some d e t a i l .  Amounts of glucuronic acid produced were measured  by means of the color produced i n ether solution with naphthoresorcinol reagent.  Intensities of colors were measured by means of the K l e t t -  Summerson photo-electric colorimeter.  The color produced by glucuronic  acid and naphthoresorcinol i n the presence of concentrated hydrochloric acid has been used to determine glucuronic acid since the test was proposed by Tollens (61).  Maughan, Evelyn, and Browne (58) modified t h i s test f o r  the quantitative determination of glucuronic acid and state that there are no i n t e r f e r i n g substances except mucic acid.  More recently Fashena  and S t i f f (62) have investigated the naphthoresorcinol test for glucuronic acid.  Pentoses, as well as glucuronic acid, y i e l d condensation  products  with naphthoresorcinol which are soluble i n ether containing small amounts of alcohol.  The addition of 2 c.c. of alcohol to 15 c.c. of ether when  making extractions of the colored compound from water, was recommended by Maughan et a l (58). As a result of our i n a b i l i t y to separate glucuronic acid i n spite of good indications of i t s presence with naphthoresorcinol, i t was decided to investigate the reaction of the reagent with substances which would possibly be produced i n our reaction mixtures.  Bergmann and Wolff (39)  reported that glyoxylic acid gives a strong naphthoresorcinol test.  The  following substances were tested i n amounts which might be present i n the dilutions used for t e s t i n g f o r glucuronic acid i n reaction mixtures. given are those produced i n the ether layer« TABLE VII. Substance  Color produced  ,04 mg. glyoi&XLic acid  purple  .05 mg, glucose  purple  Colors  TABLE VII (Continued) Substance  Color Produced  .05 rag. methyl glucoside  cloudy purple  .05 mg„ gluconic acid  none  .04 mg. t a r t a r i c acid  none  .04 mg.  none  saccharic acid  In larger quantities, t a r t a r i c acid gave a definite color reaction, as shown i n the ease of diethyl tartarate separated from an oxidation mixture using barium iodide and iodine as the oxidizing agent.  A second un-  i d e n t i f i e d f r a c t i o n separated from the same oxidation mixture also gave a good test with naphthoresorcinol.  As a result of these findings, i t was  doubted whether glucuronic acid was present i n the reaction mixtures i n which i t was Indicated by naphthoresorcinol reagent.  This conclusion was  .confirmed by our i n a b i l i t y to separate out the acid by the usual methods of procedure. (vii)  Methods of separation.  Various methods of separation suggested themselves. potassium s a l t s of glucuronic acid are c r y s t a l l i z a b l e .  The sodium and The barium and  calcium s a l t s are amorphous and f a i r l y insoluble i n water and more so i n alcohol.  The brucine and cinchonine s a l t s have been suggested, the f i r s t  by Smolensk! (35) and the second by Leutgoeb and Heinrich (40).  A question  arose as to whether i t was best to form a salt of methyl glucuronide immediately a f t e r oxidation or to hydrolyze the methyl glucuronide then to remove a l l other constituents except glucuronic acid. were t r i e d .  and  Both methods  I f present, even i n a complex mixture, glucuronic acid should  be f a i r l y e a s i l y separated.  Glucuronic acid I t s e l f i s s l i g h t l y soluble i n  - 42 alcohol.  When a water solution of the acid i s boiled, evaporated or even  allowed to stand at room temperature, water i s lost with the formation of the lactone.  The anhydride i s insoluble i n alcohol but readily soluble i n  water. In the methods using potassium hydroxide plus potassium iodide and iodine as oxidizing agents the following procedure was followed.  The  iodine was removed after the expiration of the oxidation time with basic lead carbonate and f i n a l l y with s i l v e r acetate, the precipitates being f i l t e r e d o f f . The resulting f i l t r a t e was hydrolyzed for.three hours with 2 N sulphuric acid, at the end of which time the sulphate ion was removed with barium hydroxide.  Excess barium was removed with carbon dioxide.  The clear solution was evaporated to small volume to produce a thick syrup. Hot g l a c i a l acetic acid was added, the solution allowed to cool and then several volumes of 95$ alcohol added.  A white precipitate settled out  .which was not glucurone, as should have been the case i f glucuronic acid had been present i n the solution.  The product was not i d e n t i f i e d .  Similar  methods were employed when potassium permanganate was used as the oxidizing agent with similar negative r e s u l t s . The method employing barium iodide and iodine has been described before. After removal of a l l metallic ions, a white c r y s t a l l i n e substance was prec i p i t a t e d from alcohol which proved to be the meso form of diethyl tartarate. The y i e l d of this substance was high, showing that the major part of the oxidation products was t a r t a r i c acid, and that the glucose molecule was s p l i t into at least two parts.  A second oxidation product believed to be  glyoxylic acid was not f u l l y i d e n t i f i e d . The brucine s a l t as recommended by Smolenski (35) was used i n an attempt to separate glucuronic acid from the reaction mixture.  10 grams of methyl  - 43 glucoside, 19.6 c.c. of 30$ hydrogen peroxide and 5 c.c, of f e r r i c chloride solution(.0001 gr./c.c.) were allowed to stand i n a pressure flask for ninety-siE hours.  The resulting solution gave a strong naphthoresorcinol  test f o r glucuronic acid.  The mixture was d i s t i l l e d under a high vacuum  to remove any v o l a t i l e acids produced. properties.  The d i s t i l l a t e had reducing  The residue was taken up i n water and to this solution was  added excess brucine.  The solution was refluxed f o r three hours, during  which time there was a darkening i n the color of the mixture.  After the  solution cooled, excess brucine was removed by extraction with chloroform. The f i n a l . s o l u t i o n supposedly containing the brucine salt of methyl glucuronide gave a good naphthoresorcinol test.  This solution was evapo-  rated down to dryness, under a high vacuum and the residue taken up i n hot ethyl alcohol.  On cooling, fine crystals separated out, which on re-  c r y s t a l l i z a t i o n from alcohol gave a sharp melting point of 165°C. The product was i d e n t i f i e d as <*•• methyl glucoside.  Further work on solutions  and precipitates f a i l e d to extract brucine glucuronide, which i f present must have been there in minute quantities.  This experiment proved one  thing, at least; that oC methyl glucoside i s stable under the conditions employed,  The presence of reducing substances i n the d i s t i l l a t e indicated  some breakdown i n the glucose molecule, G0H0LU3ISNS.AND.DISCUSSION. a)  Chemical method. Early work was undertaken to discover the various influencing factors  i n the oxidation of oC methyl glucoside.  Conclusions as to the amount of  glucuronic acid present were based on the naphthoresorcinol test.  Later,  when work was begun on methods of separa^ipn^and- negative-'results were  -  44  -  obtained, i t was d e c i d e d to investigate f u r t h e r the s p e c i f i c i t y o f the test.  As a result of experiments on this phase of the work, i t was  de-  c i d e d that previous indications of glucuronic acid were due to interfering substances and t h i s c o n c l u s i o n was strengthened by our repeated i n a b i l i t y t o s e p a r a t e the a c i d .  Other w o r k e r s , p a r t i c u l a r l y Smolensk! (35)  c l a i m t o have i s o l a t e d the substance i n y i e l d s up t o 30$, but our work f a i l s to corroborate these assertions.  I t i s s t f l l believed, however that  glucuronic acid should be capable of production as a result of the oxidation o f the sixth carbon atom of «c methyl glucoside.  Other reactions may go on  concurrently, a t g r e a t e r speeds than the desired one, so that yields of glucuronic acid w i l l necessarily be small.  I t i s believed that other parts  o f the glucose m o l e c u l e are more susceptable to oxidation than the primary alcohol group, so that, although this group  may be oxidized, the molecule  i s destroyed with the production of shorter chain acids or aldehydes.  The  problem resolves i t s e l f into finding some specific agent for the oxidation of the primary alcohol group.  This s p e c i f i c a c t i o n might be found i n a  catalyst and further work could be done on this phase of the chemical i n vestigation. The biochemical method seems to offer the best method to date of obtaining glucuronic acid.  The animals necessary are inexpensive both to  obtain and to keep and the yields are good. been obtained.  From dogs a y i e l d of 80$ has  Rabbits can be made to y i e l d up to 50$ of the substance.  - 45  -  SUMMARY, 1)  The b i o c h e m i c a l method f o r the p r o d u c t i o n o f g l u c u r o n i c a c i d , u s i n g  r a b b i t s has been i n v e s t i g a t e d . M o d i f i c a t i o n s i n t h e manner o f a d m i n i s t e r i n g .menthol t o r a b b i t s have been made. 2)  Procedures are g i v e n ,  The a v a i l a b l e methods o f chemical s y n t h e s i s have been reviewed and  discussed, 5)  The r e s u l t s o f i n v e s t i g a t i o n s on t h e chemical p r o d u c t i o n o f g l u c u r o n i c  a c i d from oC methyl g l u c o s i d e a r e g i v e n .  The f i n d i n g s i n d i c a t e t h a t i t i s  not p r a c t i c a l t o produce t h e a c i d b y t h e o x i d a t i o n o f a g l u c o s i d e o r o f a compound o f glucose w h i c h has the aldehyde group a l o n e , i n a c t i v a t e d , u n l e s s a s p e c i f i c agent i s found f o r t h e o x i d a t i o n o f the s i x t h carbon atom,  — — . —  0  ; 0  0 O O  — — — —  BIBLIOGRAPHY 1.  Quick, A. J . , Jour. B i o l . Chem. 97, 403, (1932),  2.  Fischer, E., Ber. 24-521, (1891).  3.  Pryde,, J . , Williams, R. T., Biochem. J . 30, 794, (1936).  4.  Quick, A. J . , Jour. B i o l . Chem. 70, 379,(1926).  5.  Quick, A. J . , Jour. B i o l . Chem. 98, 537, (1932).  6. Lipschitz, W, L., Bueding, E., Jour. B i o l . Chem, 129-333, (1939). 7.  Hemingway, A., Pryde, J . , Williams, R. T., Biochem. J . 28, 136, (1934).  8.  Fishman, W. H., Jour. B i o l . Chem. 127, 367, (1939).  9. Cohen, S. L., Marrian, G. F., Biochem. J . 30-57,(1936), 10.  Venning, E. M., Browne, J . S. L., Proc. Soc. Exp. B i o l , and Med. 34, 792, (1936),  U.  Loeb, L., J.Am. Med. Ass'n, 104, 1597, (1935).  12.  Lacassagne, A., Compt. Rend., 195-630,{1932).  13.  Burrows, H., B r i t . Jour, of Surgery, 23, 191,. (1935).  14.  Ward, H. B.,"Some Fundamental Aspects of the Cancer Problem", P. 51-66 The Science Press, New York,(1937).  15.  Boyland, E., Levi, A. A., Biochem. J . 29, 2679,(1935) and • Biochem. J . 30, 728,(1936).  16.  Willand, H., Dane, E., Ztschr. Physiol. Chem., 219, 240, (1933).  17.  Doads, E. C., Goldberg, L., Lawson, W., Robinson, R.,  Nature 141, 247, (1933).  18.  Waters, L. L., Yale J . B i o l . Med., 10, 179, (1937).  19.  Shear, M. J., Am. J . Cancer, 28, 334, (1936).  20.  Ward, H. B., "Some Fundamental Aspects of the Cancer Problem", P. 121-156, Science Press, New York. (1937).  21.  K e i l i n , D.,  Proc. Roy. Soc. (Lond)  B i l l , 280 (1932).  22.  Murphy, V. B., Sturm, E., Jour. Exp. Med., 60, 293, (1934).  23. 24.  ibid 60, 305, (1934). Fishman, W. H., Jour. B i o l . Chem., 131, 225, (1939).  BIBLIOGRAPHY (Continued). 25.  Haddow, A., Robinson, A. M.,  26.  N i t t a , Y.,  27.  Quick, A. J . , Jour. B i o l . Chem., 74-331, (1927).  23.  Bang, I., Biochem. Z. x x z i i 45, (1911).  29.  Williams, R. T., Biochem. J . , 34, 272, (1940).  30.  Williams, R. T., Biochem. J . 32, 1849, (1938).  Japan J . Obstet. Gynecol., 19, 512, (1936).  31. >:Ne'f, J'. U., 32. .33.  Proc. Roy. Soc. B 122, 442, (1937).  Ann. 357-214, (1907).  K i l l i a n i , H.,  Ber. 54B, 456, (1921).  .  J o l l e s , A. vMonatsh., 32, 623.  34.  F i s c h e r , E . , P i l o t y , 0.,  Ber. 24, 521, (1890).  35.  Smolenski, K.,  36.  Jackson,' E. L., Hudson, C. S.,  37.  G l a t t f i e l d , J . W. E., Hanke, M. T.,  38.  Craik, J . , J . Soc. Chem. Ind., 43-171 7T (1924).  39.  Bergmann, M., Wolff, W. W.,  40.  Leutgoeb, R. A., Heinrich, H.,  41.  Stacey, M.,.' J . Chem. S o c , (1939) 1529.  42.  Zervos, L., Sessler, P.,  43.  Goebel, W. F., Babers, F. H.,  44.  Goebel, W. F., Babers, F. H», Jour. B i o l . Chem., 100, 743, (1933).  Roczniki. Chemji. 3, 153, (1924). J . Am. Chem. S o c , 59, 994, (1937). J . Am. Chem. Soc., 40-973, (1918),  Ber. 56B, 1060 (1923). J . Am. Chem. Soc., 61-870 (1939).  Ber. 66 B. 1326, (1933). Jour. B i o l . Chem., 100, 573, (1933).  45.  ibid  101, 173, (1933).  46.  ibid  106, 63,  47.  ibid  110, 707, (1935).  48.  ibid  111, 347, (1935).  (1934).  49.  Hotchkiss, R.D., Goebel, W. F., Jour. 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