The Biochemistry of Certain Fungicides in the Animal Body

THE BIOCHEMISTRY OF CERTAIN FUNGICIDES IN THE ANIMAL BODY

By THOMAS EDWARD BARMAN

A Theists presented in accordance with the regulations governing the award of the degree of Doctor of Philosophy in the University of London.

partment of Biochemistry, St. Mary is Hospital Medical.School, Idomdon, W.2,

August, 1961. TO iii

ABSTRACT OF THESIS

The fates of dehydroacetic acid and of two closely related pyronee,

triacetic acid lactone and imino dehydroacetic acid have been investig-

ated in the rabbit and rat. The synthesis of (14Cid-dehydroacetic acid* 'from 14C.1-acetyl bromide is described; in addition, the preparation of

(14031-triacetic acid lactone and [ 4O4)-imino dehydroacetic acid, both from [140 J-dehydroacetic acid, are reported. By administering these labelled compounds to rabbits and rats, it was shown that [2.40J-dehydroacetic acid gave rise to about 10% 14002 in the expired air, (1403). triacetio acid lactone to 50% and Cihed-imino dehydroacetic acid to 2..3%. In the urines of animals dosed with PICJ-dehydroacetic acid, dehydroacetic acid itself, hydroxy-dehydroacetic acid, their respective imino derivatives, triacetic acid lactone, urea and two metabolites of. unknown structures, metabolites "X" and lin were shown to be present by colour chromatography and eutoradiography. Of these, dehydroacetic acid, its hydrozy derivative and metabolitelfiXo'have been iao1ated, and imino - dehydroacetic acid and imino'hydroxydehydroacetic acid shown to be urinary artifacts. Work done on rat liver and kidney slices has established that, while triacetic acid lactone was oxidised in both, dehydroacetic acid was only attacked to a detectable extent,in liver slices. The binding of dehydroacetic acid to plasma albumin was shown by paper electrophoresis.

'For structural formulae, please. see fig. 1 iv

pBEFACE,

"We feel that the aspect of the possible longterm effect of continued ingestion of small amounts of these substances (which are undoubtedly toxic in larger amounts) upon the general health is generally overlooked". (lord Kilbracken, 1961).

In recent years some 700 ohemioals have found their way into . manie food. It was, therefore, with good reason that the House of horde quite recently expressed its concern at the danger to health arising from the use of chemicals in the growing, storing and processing of food. Accordingly, the possible use of a chemical in food production should be viewed with suspicion until such a compound has been found to be without deleterioda effect on the human organism. When dehydroacetic acid was rescued from obscurity by the Doir Chemical Company and praised as having outstanding fungicidal properties, its use as a food preservative was forbidden by the U.S, Food and Drug Administration, This was thought to be necessary. because of the scanty material available on its metabolism. However, despite its ability to be absorbed AA the skin, the use of dehydroacetic acid is at present allowed in cosmetics and in certain 6 food powder preparations. In view of the above, the elucidation of v.

the metaboliem of dehydroacetic acid in the animal organism would appear to be of paramount importance. The present work was carried out in the three academic years beginning October let, l958, in the Biochemistry Department of

St. Mary's Hospital Medical School, The thesis is divided into five chapters. Chapter I- is a short. review of the literature pertaining to the chemistry and biological properties of dehydroacetic acid, triacetic acid lactone and imino-dehydroacetic acid. Chapter 2 describes the synthesis of materials used, and Chapter 3 certain methods employed. Chapter I. includes the experimental results obtained during the coUrse of this work; these are discussed in Chapter 5. I am deep]; indebted to Professor R. T. Williams for his constant advice and patience in supervising this research, to Dr. D. V. Parke, without whose generous help this work would have been impossible, and to Drs. C. King, D, Robinson and J. N. Smith for much helpful advice. Special thanks are due to Mr. F. Audas and his staff or their unstinted technical assistance, and to Miss 0# M. Parkes for typing the manuscript. The Distillers Company made this research possible by the provision of the necessary funds and samples of dehydroacetic acid.

CHAPTER 1. INrmaDucTiox Page Dehtdroseetie Acid ..,. 1 ?Wawa and chemical properties 1 Antimicrobial properties 6 .. Absorption and distribution 12. Toxicology 4,4o, * -Ars * op ** ors... ******** ***lie** 13 Chemical Pathology ....,....,...... ,.... * .. ** edloalip 16 Detoxication Op * Ora * aaelpaeaaalkoo ** a ** afol#04,001 ****** 11 * a *** isse 17 Triacetic Acid Lactone ...... ********** p.1"...... , ************ . 19 Physical and chemical properties ...... -...... ***** • 19 Biological properties ,...... ****** ....,..... 21 Zaino dehydroacetic Laid iliaaroolvorerfilloOlkailbeira * iii * a * Oa ***** gait 23

CHAPTER 2. MATERIALS Syntheses of [140-Compounds * 25 Dehydroaeetic acid **** iva.aairallife00164041,1,* * eala **** 25 Triaaetio said lautone ****** 410....tesartrolosot•toloottoolos 29 'mina dehydroacetic acid faaaa.a0 **** 00.4itairodia **** **** 30 IMAM, Materials *** ** 30 - Atteepted Syntheses of Certain Pyrones ****** 31

CHAPTER 3. METHOD Metabolism %Ober 37 Estimation of Respiratory Carbon Diodde * ** 38 Chromatographic Methods * Aloreesosiorsmooss 39 Isotope Dilution Experiments ***** 0... Moat ** ** oosoorole, 39

CHAPTER 4. THE METABOLISM OP DERIDROACETIC ACID AND SOME RELATED PIRON&S IN THE RABBIT AND RAT. Chromatographic Investigations of the Urines of Animals administered certainpyronee ..,...... The Isolation of Metabolites from the Urines of two Rabbits administered Dehydroasetic Acid 51 Identification and Properties of Hydroxydehydroacetic Acid is. 53 Some Properties of Metabolite 1.I" laalitilsoolarafaifkoaaasaataikaaa 58

Page T Metabolism of [14CAJ.Dehydroacetic Acid, (1403,14riacetto Acid Eastone and i1404J-Imino debydroacetio Aoid in the intact Rabbit and Rat 58 The Conversion of Dehydroactetio Acid and Rydroxydehydroacetio acid into their respective Imino compounds in Rabbit and Rat Urines 4 ***** 44 ******** 10444•444.4 60 Tissue Slice Experiments 10,11P000 **** ** 71 The Plasma Binding of Debydroacetic Acid

=AMR 5, DISCLIZION 89

APPENDIX I. Physical Properties of Certain Poems ...... 104 APPENDIX II. The Rates of Excretion of 14CO2 lathe Expired Air and total Urine Radio. activities in Rabbits and Rats sed on [140A)-Dehydroacetic Acid, ., 3Triacetio Acid Laotone or [40.4 Imino dehydroaeetio Acid 110

APPENDIX XII', Diets. 00,4104,.0,00.0041046 *** * 001004. 00040404001$ 120

REFEREWES *** 4 * 404,4 121 CHAPTER I

INTRODUCTION

DehYdroacetlo Acid (a) ANVARELVand Chemioal Properties Although dehydroacetic acid was discovered. as long ago as 1866 laeutber), it was not till almost a hundred years later that convincing evidence of its structure was presented (Berson, 1952). Since the turn of the century — when the compound excited the interest of several eminent chemists (Perkin, Oswald, Collie, Hilditch) — there has been a dearth of information on the chemistry of the pyrone in the literature. The first edition of Beilstein unwisely included dehydroaoetic acid in the aromatic. series, since when it is heated with barium hydroxide (Collie, 1891a; Collie and MTer.4K, 1893) orcinol is formed One might equally well argue that acetone is aromatic since it is polymerized to mesitylene on treatment with sulphuric acid! Debydroacetic acid when carefully purified by sublima— tion is a_colourless crystalline substanoe without odour or taste. It dissolves readily in acetone, benzene and hot ethanol, but is much less Soluble in carbon tetrachloride or cold alcohol. Its solubility in water is low (<100 mg./ 100 m1. at 25°) but the pyrone dissolves readily in cold aqueous alkali to give salts. The acidity dehydroacetic 2.

acid (pK 5.3) is due to its enolio character; consequently it gives a blood"red colour with ferric chloride and decolourises bromine water. The 2,4Apyronone nucleus of dehydroacetio acid is reser. kably stable to hot mineral acids. Concentrated hydrochloric acid converts the compound into 2,6.0dimethy140-pyrone (Collie, 169I8) but At sulphuric acid results in deacetylation to triaoetic said lac:tone (Collie, 1891b) Whilst 83 sulphuric acid provides an isomer, namely 2,6vdimethylorone..3.- carboxylio acid. (Collie and liilditoh, 1907). Dellydroacetio acid is steam volatile but some decomposition to 2„6.dimethyl....

4.pyrone and carbon dioxide wow% (Collie, 18910)* Dehydroaoetic acid is very susoeptible to the action of strong alkalies; acetone, mriiinn c acid, acetate and carbon dirrilde•have been mentioned as products of allsOi decamposi.. tion (Perkin, 1885 1887)• The pyrone reacts readily with he usual ketonic reagents yielding crystalline derivatives. Thus, an oxime and a phanylhydresone are known (Perkin, 1887); the latter undergoes oyalisation when treated with hydrochloric: acid (Benary 1910):

Ph— Nil— N 1111 N tai

G 0— 3 3

ill

The act of ammonia on debydroacctio acid is rather

ourious In the cold, or on gentle waraixig, an tid.2710 derivative is foraged. (Ockl.lie, 1894. Refluxing with strong ammonia yields 2j6odisetbyl.4*.:pyridone..3.carbowlio acid (Collie ,and

IUlditoh, 1907; Rassweiler and Adams 19214. The latter is

readily de carboxylated when heated to 27CP to give 40.1utidone (Rasswsiler and Moms I9210• 4.

0 0 NH

0— OH NH3 3 cold.

0 OH 3 triacetic acid 3.aotone

refliut, NH 3

2,6.dimethy1q.4..pyridone.- 3-oarboxylio acid.

40.1utidone

one slmAlswiY a in the cold. and at pkytiiologioal tH to form Zch b es (Iguchi and Hisataune, 1957; Id4ohi, Hisateune, Himeno and Nuraoka, 1959).

Dehydroaceti© acid. may be reduced to deeoxydehydroacetio aoid, 3-et4y1../...hydrov..6.,methyla.5.6.dikydro..2-pyran and 3satootY1-40-hydrov.6..met1yl..5,6.413hydro..2• 3yran, The effects Of different reducing agents is summarized below: (1C) Pdfcharocal "IP Gil 800 40 lb. pressure 3 (Walker,, 1956)

3 -ethyl-lehydrozy-6. raethyl..5 a 6—dihydro-2•• pyran.

desoxydehydroacetio acid

1).; (Collie and Le Sueur t 1894) (Mn lacand Wanozura, 1933 ) •

(Bart'els.Ceith; and Turner, 1960)

5»acety1•44ydroxy4scothyl 5 #6-dihydro-2,4vran.

Debydroacatic acid reacts with a x laer of aromatic aldehydes to form 5•cinnatacyl-lishydroxy,06«methy1.2-pyrones:

0 0 3 These acepounds are highly coloured and fluorescent (Wiley, 6.

Jarboe and Ellert. 1955). The existence Of a monobromide of dchydroaoetio acid was shown by Perkin (1887) who prepared a (=pound of empirical formula C8H704Br by refill/tog the pyrone with bromine in chloroform. Phosphorous pentachloride reacts vigorously with dellydroacetic acid even when diluted with the oxyohloride to give a compound of composition 06H602012 which on treatment with sulphuric acid affords 2,6.dimethyl..4,Tyrone.3.0arboxylic acid (Collie, 1891a). Both methyl and ethyl ether3of dehydroaoetio acid are known (Parkin, 1887; Collie and Le Sueur, . 1894). The methyl ether is probably 6.methy1.3.acety14. methoxy04.pyrone since it dissolves readily ion water. (b) AntimicrObial,Proverties 1110cman„ Brian and Hemming (1948) were the first to report on the exoellepoe of dehydroacetio acid as a fungi. static. This observation was followed by the experiments of Coleman and Wolf (1949) and Wolf and Westveer (1950) who showed the compound to have a wide antimiordhial spectrum indIUding fungi, yeasts and bacteria. However, workers such as Brodersenandlciser (1946), Seohaut (1952), von Schelhorn (1952) and Rausoher card Ardelt (1954) regarded dohydroacetio acid as a poor baoteriostatio (see Table 1). Ingram, Ottawsy and Coppodk (1956) compared the compound unfavourably with benzoic acid in this respect but found that it arrested the growth of yeasts with equal ability and exceeded the potency of the aromatic acid as a fungistatic. The evidence for the fungistatio properties of dehydroacetio acid is overwhelming (Nagasawa and Kawakami, 1952; Shimose, 1952; di Caro, 1953; Okabagashi, 1953; Ozawa, 1953, Urakabo, 1955) and the compound has been used as a preservative of wine (Nnehida and Taki, 1955; Sudario, 1954), of latex (Resing and de HaansoHomans, 1953), of leather (Hausam, 1954), of artificial silkworm food (New Scientist, 1960), and as an improver of seed germination (Hale, 1954). Other applications of the compound include its use as a mosquito repellant (Smith and Burnett, 1949) and as an antituberoular agent (Kunigoshi, 1958). Foods rich in fats and protein have shown excellent keeping properties after add4tion of 0.01-0.5S (w/w) of dehydroacetio acid, and 2r4 in bread wrapping has been found to be as effective as 7i. of benzoic acid (Ingram et al 1956; Beehaut, 1950). However, the compound appears to have a possible oxidative effect on foodstuffs since Tarr, douthcotti and Bissett (1950) showed that fish preparations containing large amounts of the pyrone had excellent antibacterial properties but became rancid after a short time. The co and was shown by Fosdick, Calandra, Blackwell and Burrell (1953) to have some aweless in the treatment of dental caries. Of 380 compounds tested, only dehydroaeetio acid and sodium 11.3.auryl sarcosinate were effective at 5 mip. per 100 ml. in mouth. washes, Both compounds were adsorbed onto the dental plaque. Were it not for its toxioproperties, therefore, dehydrow, acetic acid would be en exoellent food preservative (Mgr= et al, 1956, Ingnam, 1959,-8atimes-s-1969). Its high pK value of 5.3 ensures the presence of the =dissociated form inmost food*. stuffs andingrameala(1956)Siuggest that weakly acidic food preservatives such as benzoic and sorbio acids are only fully

as tive,Whin present in their undissodiated form (also see Grove, 1940. Since the toxic properties of.dehydroacetio acid are said to be comparable with those of carbolic acid (Uartin, 1957), the use of the acid as a food preservative is now forbidden in most countries, The exact mode of act dehydrosoetio acid in arresting the growth of microorganisms is a moot question. The anti- miorobial properties of numerous a, ...unsaturated ketones and unsaturated laotones are well known (Rablin, 1946), Geiger and Conn (1945) suggested that these °harm:teas Jos were due to their ability to react with the sUlphydryl groups of certain essential enzymes. Thin theory was supported by 0avallito and Haskell (1945) who showed several unsaturated lactones to react with oysteine to give stable amphoterio prodUcts, Later, Brodersen and. Kjmer (1946) allowed that the baoteriostatio action of several unsaturated 1/acetones was abolished by cysteine. However, doubts were thrown on the importance of the unsaturated laotone structure to bacteriostasis on finding that compounds such as triaoetio acid lactone and totronic acid were without biological activity, Severe], workers have shown that the a,f3.unsaturated ketone •structure is essential for the activity of a large number of bacteriostatios (Marrian, Russel and Todd, 194.7; SohraufatItter and Bernt, 1949; Binderkneoht, Ward, Bergel and Morrison, 1947). As in the:case of the unsaturated laotones, oysteine was found to abolish antibacterial activity. Geigy (1948) showed that 2.4ydroxy.wylophenone inactivated sulphydryl enzymes such as auaoinic-, alcohol. and triosephosphate dehydrogenases, and also urease, This activity was abolished by thioglicollatei Dehydroaoetio acid may be regarded as both an unsaturated lactone and an ad-unsaturated ketone and might therefore be expected to inhibit sulphYdryl enzymes generally* Seevers,. Shideman, Woods, Weeks and Kruse (1950) showed that this was indeed the ease as regards sucoinic dehydrogenases but the ccmpound was without effect on pepsin (Eeohaut, 1950) and it aotivated urease (Seaver. 1950). Purthermore, its inhibition of aucointo dehydrogenase was not reversed by the iddition of cysteine Or glyoollatei nor did it react with oysteine (GaWallito and Haskell, 1945) Arnatein and Bentley (1953) have suggested a mechanism 10.

by which certain microorganisms convert glucose into pyrones. Since it is probable that the pathways involved in the forma. tion of pyrones an& shikimic acid are closely. related, Ehrensvgrd (1955) suggests that the inhibitory effeot of dehydroaaetio acid is due to its interference with aromatic biosynthesis.

Table 1. The Antipicrobia1 Activities of Pekvdroacetio Aoid, (a) Comparison of the fumgistatio activities of dehydre. acetic and *orbits acids (pg/M1. for complete inhibi. tion). From MoGowan.a.ele. (1948). Debydrosoetio,hoi4 Sorbio Acid Fumarium gramiz erw 80 62.5 Peniailliwn digitatum 400 400 Botrylis allii 50 4.00

(b) Comparison of e,af offec tsdehydroaoetio acid and benzoio acid on (sertai n yeasts (Yerceotage °omen.► tration arresting growth). From Beohaut (1950

Dehydroacetic Aoid Benzoic Act;

Sacoharopyo nerevibia 0.025 0.5

Aerobaoter wrogenee 0.3 0,25

(contd.) 11.

Table 1 (contd.)

( 0 ) The effect of dehydroacetio enid. on certain microorganisms (minium percentage conoentration arm.. ting the growth of the dormant form). From Rechaut (1950). ethericiet cola. 0.40 Alcaligenasfeacialis 0.40 Salmonella typhosa 0.20 Aspergillus niger 0.05 Penicilliam =pans= 0.01 Rhizopus nigrioans 0.05

(d) Th9 antimicrobial activity of dohydroacetic acid minima percentage aonoentration which inhibits). Fr= Wolf and. Westveer (1950).

ilerobaater Oa rogenee 0.30 iliorocoocus pyogenOs 0.30 Salmonella typhoea 0.20 ligitatum 0.03

Rhizopus nigricans 0.04. Sacoharomyces cerevisite 040

The growth of Panic, ilium urticre is 1000 xibited by 0.002-0404'1 del‘ydroaoetio acid (Ehrensn4irdi 1955). 12.

(a) Alsorntionandyistribution

The rapid absorption of dehydroaoetic acid from the gastro'.

intestinal tract can be predicted from the work of Brodie and

Begben (1957) and Schanker (2960). The wrone. - being weakly acidic - would be rapidly removed in its undiesciliated form fran.the acidie stoMach contents leaving small amounts to be absorbed from the small intestine and colon. Thus, on applying

the formula of Schanterv it can be estimated that the ratio of the concentrations of dehydroacetic acid in.theplasma and gastric juice is 200:1. The sodium salt should be equPy.well absorbed. The above is supported ter Wbods, Shideman, Seevers, Weeks and Kruse (1950) who found both dohydroacetic acid and its salts, to be rapidly absorbed in man, monkey, dog and rat. In the dog, for example, only 5% of the orally fed compound was recovered in the fasces after a period of one week. Neither ethanol nor olive oil were found to enhance the absorption of dehyciroacetic acid. In common with most lipid soluble drugs, the undissociated acid probably passes across the epithelial lining of the intestinal mucosa by simple diffusion through aAlpoid-sieve -type Wundary rather than by active transport Dehydroacetic said is rapidly absorbed through the skin, the sodium salt inch less so, No selective distribution or accumulation of dehydroacetio acid occurs in the liver, small intestine, colon, cerebral oar or hemisphere, cerebellum And medulla, skeletal-0W cardiac muscle, lunge, stomach, spleen, kidney or bile in the dog. Analysis of the dohydroacetic content of the blood of dogs fed on the pyrone shows that the plasma contains 2•22 times as much as the erythrocytes; in man, this ratio is only 1.76 (Woods et al. 1950). It is possible that the lower plasma level in man is explained by less plasma protein binding This would explain the greater efficiengy in elimination of the fungicide in the human (Woods 'jg. 1950). (d) Toxipology Ingram Djuhle (1956) and Barnes (1959) regard dehydroacetio acid as too toxic to be of any use as a food preservative and Martin (1957) compares its toxicity with that of carbolic acid. Shideman gta. (1950) however, had previously suggested that the pyrone had low protoplasmic toxioity to mammalians (see Bell, Tables 2 and 3). Of the species studied, the dog is the most sensitive to debydroacetic acid, whilst the mouse shows the greatest resistance to the drug. Three human subjects have tolerated 10 mg./kge. May for 115 dws without ill effect. For shorter periods hospitalized patients have carried plasma concentrations as high as 30 mg./100 ml. without any evidence of'toxicity (Shideman, giaa. 1950). TABLE 2 THE TOXICOLOGY OP DERYDROACETIC ACID

1 • i 1 1 ; Node of Toxic Symptoms Pathological Changes References Species Administration 50 9 , v ,

Rat oral lg./kg. 1.92 g./kg. emaciation contracted steam* Spencer, Rowe wad with blood-tinged MoCollister (1950) contents. Mouse intxapexitoneal 1.18 - - - Shidefman, Woods injection and Samara. (1950) Dog oral 0.4 - Salivation, Seaters, Shideltan, 0.25 retching and Woods, Weeks and vomiting, ataxia, Kruse (1950) incoordination, weakness, stupor, musolo twitching, tonic, and clinic' convulsions followed by daith.

Monkey oral - as for dog inflammation, or Spencer et al, the small (1950) intestine. Takla 0ronto to4citv of 44Ydroacetl, api4

Maximum HaximUm Species tolerated Period tolerated daily Daily dose Referencoo daily dose dose with oaus death (sgilkg•-) forced feeding g.) (iiiikg.) Rat .0.1 2 yrs. 0.2 0.3 Spencer et al. (iBlir

Monkey 0.1 1 yr. 0.2 0.3 Spencer et al. (155,57- Dog 0.05 200 days 0108 0.2 Seevers et al. (1950)

Debydroacetio acid was found by Spencer 21g. (1950) to be without effect on the human skin. Death vas caused when applying

5 gm./kgm• to the skin of rabbits; 3 gm./kgs. merely resulted in a transitory loss of weight. The toxic symptoms of dehydroacetic acid are indistinguishable from metabolic acidosis. This increase in, acidity is due not only to the ;pone itself but also to acidic residues resulting frog inoomplete intermediary metabolism (Shideman, &La., 1950).

Sodium barbital (at 0.2 gm./kgm.) was found to be the only effective antidote against dehydroacetic aoid in the dog. Base

yielding salts, glucose and vitamin concentrates had no effect in

alleviating the toxic symptoms (Seevers, 916ja. 1950). (e) 911011434 iath°4117 In oommon with the other auccinic dehydrogenase inhibitors, dehydroacetio acid affects the tubular secretory mechanisms

requiring energy from the Kreb'e cycle. Thus, Shideman and Reno (1951) showed the pyrone to suppress the tubular secre- tion of certain acidic drugs (e.g. peniallin and phenolsuiph- thalein) and basic drugs (e.g. huniet4r1 nicotinamide). Both of these mechanisms require as a source of energy certain 'tufty rich phosphate compounds (Bwer, Painter and Weibelhaus, 1950). It is possible, therefore, that the toxic effects of dehydro. acetic acid are due to the retention of certain toxic substances - in addition to its effect per 44D. Dehydroacetio acid does not affect the renal blood flow, glomerular filtration rate, nor the tubular reabserptive capacities for glucose or phosphate (Shideman and Rene, 1951). This is in keeping with the Suggestion of Beyer sajga (1950) that succinate oxidation is not intimately associated with glucose and phosphate absorption. The experiments of Weeks, Chenoweth and Shideman (1950 ) suggest that-dehydressetic said has an effect on the animal organism other than its inhibitory action on saccinicdehydro. &mettle.' These workers showed the fungicide to interfere with the spontaneous contractile activity of the isolated rabbit rT

intestine by sodium aoetate whilst malonate (and triacetic acid lactone) had no effecU Although the toxic symptoms of dehydroacetic acid suggest an interference with the cholinergic mechanism, the drug did not inhibit oholine esterase (Seevers, st.d.,- 1950). Other enzymatic activities of the. pyrone include its complete inhibition of the acidltoephatase of olives, but its stimulation of the, alkaline phosphatase of tomatoes (Bertram and Mosquera, 1954). Dehydroacatic acid is without effect oh the digestive enzymee (Osawa and Kagaraki, 1952). A possible effect of dehydroacetie acid on the biosynthesis of aromatic compounder in F00.0illi* tillicApar has been noted elsewhere (n0). In 'View of the above, it it rather. odd that Barnes (1959) records the fungistatie as having no known effect on any enzyme system or to interfere with cellular metabolism. (f) Petoxication That dehydroacetio acid is extensively degraded in the animal organism.is suggested by the experiments of Shidecan ILA. (1950) who showed,that the dog eliminated only lc% of the prrone within the first 4, of administration, followed by 5-10% during the course of the next two dep. Thereafter very little was excreted. This indicates that 80-85% of the compound is metabolised in the dog. Similar results were obtained with monkeys; in man, however, almost twice as-much • was found to be eliminated by the renal route. There is no - evidence that kidney excretion is modified by-the prolonged administration of the compound (Shideman sila"1950)i That the liVer is involved in the detoxieation of dehydro. acetic acid ie'suggested by the fact that liver damage.in mice increases the toxicity ofthe compound. The Slow excretion-of debydroaoetio acid can be explained by its ability to combine with the plasma proteins —and is. . therefore unavailable for glemerularfiltration and by tubular reabsorption.- Woods x..(1950) showed the binding of debydroacetic:acid•with plasma to be inversely proportional to tne:amountOf•pyrone present. ?or example in the dog, the maximum_binding.was reached aV70.100mgm./100 ml4 at higher concentrations the amountorbinding decreased. A peak plasma level was reached after administration; this was -followed by a slow decay ..the drug being present, in detectable amounts in the blood as long as four days after Administration.- -Woods 91.12146 (1950 found dehydrogcetic acid to be present as long as three veeks.after administration in human Plasma.. When given intravenously the drug showed a similarly olow plasma decay level. So far as the fate ofdebydreacetio acid inthe animal 19:

body is concerned, little is known. Tests for acetone, acetoacetate and reducing Eubstances in the urines of the

dog and man receiving the drug were consistently negative

(Shihimangl_pa., 1950). As only small increases in detectable dehydroacetic acid were found after acid hydrolysis, conjugation is thought not to occur to any appreciable extent. Barnes (1959) suggests that the compound La broken down into

various acids which are then oxidised via the Krebts cycle to carbon dioxide and water.

The in vitro experiments of Shideman 2/..a. (1950) yielded no clime as to the degradative pathways of &hydro.. acetic acid: rat kidney, liver and cerebral cortex slices

and skeletal muscle minces showed no decrease in added pyrone concentrations even after prolonged incubation periods. Meister (194§)similarly obtained negative results with rat liver and kidney slices and homogenates. DeWfdro- acetic, acid was also found to be nnsffeeted, by a lactonase causing the cleavage of the pyrone ring of trincetic acid lactone to triacetic acid. Triacetic AO& Lactone (a) FhYSAcal and Chemical Properties Triacotic acid lactone was first prepared by Collie (1891b) who showed it to be the deacetylation product of dehydroacetic acid. It is a stronger acid thOdehydroacetic acid and as In the case of the latter, its acidity is due to its enolic 20.

character. The lactone is stable to concentrated sulphuric acid up to 2000; hot dilute acids or water decomposes it to acetylacetone and carbon dioxide whilst boiling alkali gives acetic acid, acetone and carbon dioxide. It is thus more stable than triacetic acid which decomposes rapidly in aqueous solution at room temperature (Witter and Stets, 190). With bromine, triacetic acid lactone forms amonobromide- 3-Pbromo4riacetid acid lactone - but nhosphorouspentachloride .produces a resinous material (Collie, 11391W. A nitrate is .formed by the action of a mixture of concentrated nitric and' sulphuric acids (Collie, 1891b); this is reduced by tin and hydrochloric acid to 3-amino4riacetio acid lactone which with nitrous. acid affords a diasoanhydrias (Arndt and Avan, 1951.). ForMic or phoOphoric acids have little effect on this compound but the action of 30% sulphuric acid results in extensive degradation. 0 0 :nitrating mixture

C14, 0 triacetic acid 3-nitro-triacetic lactone acid lactone 0

012 ei42 /%0 .=,3-amino-triacetic acid lactone 2i

Two met4y1 others cf tric,eetio acidlootone are lirm,!no. t% elyf 6.6neth7l-4-oethOly-2-PYrone and 6.43sth71-acetialv.4- ;wow. Troth are proluced the action or dissomethane on the plrone (llestOttiorni Gottlieb and tlferansit 1;59; Bnelock a LAtho 140;- and Cieslook.- Lank and Cbmiehewa, 296G). lethyle. tion wi th methyl eulObato affords only 6-methy1,02..7otho:v.A. tyrono (BuiLoCk and SAth 1960; the action of methyl Wide on the silver ealt or trisestio mid /Acton. yields virt is- probably again -a mixture .of the to (Sproxton,' 1?06)- Wiley and Jarboe t19564 coupled twelvs 1 diazonion talc with the lactone to gig aryl kermess. /base roanmy rearv:Alge n treatment with either sviosOus acid or base to 1..aryl..3.oarbolr- 1../rwridazonee:- 0

bj 4 1or1sal ero TrisOotio acid laetone is apparently wi activity. (rodereen and K3sev,1946i Shtleman, 195W); in foot Ehrenevird (1955) showed it to proaote the nth or Zenlnilliona, prticaee. The coupon:0 does not inhibit am, dehydrogenase Cgoevers, 91.a. 1954 With an I.D5 of 4.34 in mice it is much lees toxic then dehydroacetio acid

(Shideman 21, .11,,, 1950).

The biological lability of triacetic acid lactone was first

noticed by BreuadaACUlusoy (1947) Who found that the compound gave

rise to acetoacetate but not acetate in rat liver homogenates. A year later, Witter and Mote (19e8) showed the lactone to be meta—

bolised at only one eighth the rate of triacetic acid, and it wao

suggested that the diketoacid was an intermediate in the formation of acetoacetate. ?Meter (1949g,and 1) then showed by his elegant opectrophotcmetric experiments that triacetic acid lactone is

opened to triacetic acid which is enbeeepently hydrolysed to

acetoacetate and acetate. Both liver and kidney homogenates of the rat were found to catalyse these two coneequetive enzymatic proceesos; the lactonase was more active in the kidney than the

liver but triacetio acid was hydrolysed more rapidly in the liver, The different pH optima of the two steps suggests the participation of at least two enema. The lactonase and hydrolyase were found by high speed centrifugation to be located in the deposit and supernatant, respectively. ' Neither the lactose nor triacetie acid were found to be destroyed at appreciable rates* homogenates of the pancreas, spleen, skeletal muso1A or brain. Beef liver (Witter and Stote, 1941) and cat kidney (Meister, 1949a) did not destroy triacetic acid lactone to any detectable extent.

stIMari of the Metpholiem of Tr1acetiq Acid Lactone

nlactonasew •optimum pH 5.9-6.3 3 triacetic said triacetic acid lactone itfydrolase optimUm pH 7-7.3

CH CH 2 3 0 () 0 CH 3 OH

acetoacetie acid acetic acid

;m10414rdroAttetio A01.4 The dearth of information in the literature on imine-dehydro. acetic acid is extreme. It was prepared by Collie (I89)and v. Pschman and Weiler (1,893) "and its: constitutien was ahown by Feist (1890) to be 3—acetimideyla-triacetic acid lactone. It is farmed by the actiencf ammonia on dehydroacetio acid, and is decomposed both by acid and alkali to ;ire the original prone and ammonia. From the experiments of Wolf and Weetveer 50) it is clear that lainelektrdroacetis acid ham only foeble antimicrobial activity (nee Table 4).

Table 4. Comporison of the antimicnrobial activities of debilroacatic acid and its laino derivative Oaf and uestvear, 1950) Debyir›. dehydroaoatie acetic acid said Aerobacter memo- nee 0.30 0.30 Ilicrocoecun atomise 0.40 .z1.30 Salmonella taboo* 0.30 0. PontaIlium digitatom 0•30 0.03 Rhisopus nit-rig/ins 0.20 0.04 Saccharasyces corevielas 0.30 0.10

(the figures reprise*nt mtraisam pereentaqe e-neo ntre tines irdd.bit) CHAPTER 2 MATERIALS

theses of rut Jammu,

t150.0,0ydrmiostie 40id Atom ethyl acetoacetate. Dekidroseetic acid vas first prepared by Geuther 486E4 who passed carbon dioxide over the heated sodium derivative of ethyl acetoacetate. Although Perkin (1887), Collie (1891g) and Oppenheim and Preoht (1893) had prepared the pyrone by the condensation of ethyl acetosoetate at high tempera.. tures, it was not till much later that a reasonable yield vas obtained by this method. Thus Arndt (2955) obtained a yield of 53 by refluxing the ester ter several hours in the presence of a trace of sodium hydrogen carbonate. In the present work it was necessary to eynthesise [1,3-240 ]- 041 acetoacetate since this compound was not ammeroially able. The ester was prepared by condensing [2".140j.ethyl acetate in the presence of sodium (Inglis and Roberts, 1948); [1-240). ethylacetate, in turn, was prepared by refluxing anhydrous sodium [10.140.acetate with trietbyl phosphate (kopp 1950). In prelimi nary experiments using inactive acetate, overall yields of 15..20% more obtained; however, with the 24C-oarboxyl labeled acetate, less than 0.1% of the original activity was recovered as [140- dehydroacetio acid. This method was abandoned. Irmasotrl brcaidg. Collie (1891g) reported that when acetyl chloride is heated with pyridine, small amounts of dehydroacetio acid are formed. Sauer (1947) found awl halides to be readily dehydrohalogenated in the presence of triethylamine and prepared diketene from acetyl chloride by this means. The polymerisation of diketene to dehydroacetic acid has received considerable commeroial interest; basic catalysts such as sodium phendlate, podium hydroxide, sodium ethoxide and trietbylemine (Steele, Boos. and Dull, 1949) and sodium acetate (Abrdt, 1956) have been used in the dimerisatton. In the present work, [140}4410hYdroaootis acid was prepared by polymerising [1.. O1-acetyl bromide in the presence of triethylmaine. (4- 40-acetyl Chloride was not available). Before using the 140-carboxyl labeled compound, preliminary experiments were carried out to find the optimal conditions (see Table 5). Experiment No. 5 was finally' adopted for the radioactive synthesis. TABLE S. RENTAL CONDITIONS FOR THE aEPARATION

OP DEHTBROACNTIC ACID PROM ACETYL BROMIDE

Time for Time for the Weight Experiment Weight Weight acetyl bromide the dikeittne Yield der acetyl d ethylamins trietheariOne to dimerise to dimerise (reflux) t 4.14; 10 min. 0.72g 20 min. 1.5% (reflux) 2.5 2.05 2 hrs. 1.44 2i hrs. 6% (reflex) 3 2.5 2.05 overnight 1.44 .2 hrs. 15%

2.5 2.05 overnight t.44 . 2 bre. 1%

5 2.5 2.05 overnight 1.44 2 bre. 17%

2.5 2.05 overnight 1.44 2 hrs. 17%

NOTES: 1. In all the experiments benzene was used as solvent (10 :amount acetyl bromide) except in one case (4) where acetone was employed. 2. In experiments 5 and 6, the triethylamine Iverobromide precipitated during the course of the preparation of the dikeXttne was removed by filtration before the addition of the second lot oftrietbylamine. In experiment 6, the benzene—dikeNine filtrate was concentrated under reduced preasurs to half its volume. This was followed by the seoond lot of triethylamine. The synthesis of 1-Dehystructfottukciet (3411140j acetyl-

&methyl-2p 3i.dthvdro [2 6"11t3kayran-2,Irdione).

•• (011 triethylamine 2 CH= 3.60.13r) 2 18 bra. 0 •=CH (diketene) triethylamine reflux 2 bro.

C 0 H\ /•\ II G 0— 0 — CH •IIa• •01 • 3 \o OH 0 (dehydroacetio - acid) 3 I Dohydroacetic aotd labeled with 24C in four of its carbon atoms vas prepared from [1..- 2401 acetyl bromide (Radiochemical Centre, Amersham). Anhydrous benzene (25 ml.) vas planed in a dry 100 m1. flask fitted with a magnetic stirrers condenser, tap-funnel and anhydrous 0412 guard tubes. [1.140] Acetyl bromide (5 g., 1 mG) in 25 ml. dry bensene was new added to the flask followed by a solution of freshly distilled triethylamino (4.1 g.) in dry benzene (20 ml.) with vigorous stirring during 1 hr. The flask was then stoppered and left overnight. The triethylamine hydrobromide which had separated was removed by filtration and washed with hot benzene (2 z 10 ml.). The clear brown filtrate was reamed for 2 hrs. with 2.05 gm. triethylamine and the solvent removed by distillation. The tarry residue was then coaidietilled with 5 Sm. Trigol (triathylanegly001, B.D.H. Ltd.) at 4 mm. pressure and 120.00°C. A nrikold trap containing 5 ml. anhydrous acetone yeti placed between the condenser and vacuum pump. Crude dehrdroacetio acid (m.p. 103.50, yield 493.4 rags or 28•3%) aeperated in the distillate. This was collected and reorretallized from ethanol and finally C014 The pure [ 4DJ-dehydroacetin acid had m.p. 108-9° and a specific activity of 519 twig. The recovery of lAC was 18% based on the [1-140j acetyl bromide used, and a further 0.h% of the 240 was recovered as dehydroacetio acid by working up the residuee. t%J[Triacetic Acid Laotone., Emeethyl.e2 3ftdihydro [2,4,6.0403j pyran-2dedione). [1404)-dehydroacetio acid (208.3 mg. containing 29.3 to 140) was heated to 130-.135° with 0,5 ml. 90% (v/v) H2604 until evolup was cooled and poured tion of CO2 ceased (15 min.). The mixture into 2 ml. ice cold water. The triacetic acid lactono which separated was filtered off and recrystallieed (charcoal) from water. The product had m.p. lag° (yield 51 mg. or 30.6%) and a total activity of 7.17 pc or 140.6 po/g. 2L1 4.....iinDill sgazi o. (341-140J acetimidoy1-6...mothy1- 2,3Klihydro [2,4,6-1403)-pyran+2,4rdione). [1404)Dehydroacletic acid (23 mg. containing 11.90 pa 14C) was treated with 0.5 ml. 36$ (w/v) 163 and the mixture left overnight. The solid mesa was filtered off and dried over anhydrous M004 in vacuo to give 20 mg. (89%) of the crude imino compound. ffnit. 301.0nactive imino dehydroaaetic acid was added and the mixture crystallized from methanol and then from ethyl acetate to give the pure compound of m.p. 208.5 - 209° (yield 221.3 mg. or 59%) and a total activity of 6.92 pa or 31.29 po/g. Inactive Material, DOhYdroacetio acid (3-acety1.4..Mathyli.2,3-dihydropyran-2,44ione), m.p. 109° (Distillers Co. Ltd.), was purified reorystallisation from ethanol and carbon tetrachloride. Triacetio acid 1actons (6-methy1.2,3-dihydropyran-2,4-dione),- m.p. 188°, was prepared according to Collie (1891p) by the action of hot 90$ sulphuric acid on dehydroacetio acid. Fran Imino-dehrdroacetio acid. (3-acetimidoy1-6-methyl-2,3-dihydr%.2,4- dione), m.p. 208..9°, was prepared by the action of ammonia on dehydroacetic acid (Collie and )$ers, 1893). . Depoxr-dehydroggetio acid (3.methy1-6-methyl-2,3-dihydropyran- 2,4-dione), m.p. 185°, was prepared by the catalytic reduction of 31.

dehydroacetic acid (Malaohowski and Wanozura, 1933).

A6*Dimqtbin-40-pyr9np, m.p. 132.5° was prepared according to

Collie (1891g) by refluxtng dehydroacetic acid with 10N-H01. 2.6-Dimethyl-ho.natrque13-cartoxylic acid, n.p. 99°, was formed by the action of hot 85% sulphuric acid on debydroaoetio acid (Collie and Hilditch, 1907). Attempted Sinthopee ogr Certain Pironee TgXacetio Acid Ifactone-3-oarbovilic Acid (6-methy1-2,3-dihydro- Wranp2,4-dione-3..carboxylic acid). .

When compounds containing the group C11iC0- are treated

with alkaline hypohalite solution, oxidation and rupture of the carbon-carbon bond may occur. Accordingly, attempts were

made to oxidize the 3...acetyl side chain of dehydroacetio acid

by this means, Thus, debydroacetio acid was dissolved in either dioxano or 5% sodium hydroxide and an aquas solution of sodiva hypohalite (hypoiodite, hypobromite or hypochiorite)

added in the los cold and the mixture;- vigorously stirred. Chloroform extractions of the acidified mixtures failed to yield any crystalline compounds other than dehydroacetio acid (which was recovered in 5040% yield) and a non-acidic oily material. Chromatography of the reaction mixtures showed dehydroaoetio acid to be the only acidic substance present (using bromooresol green as detecting reagent). 32.

341-krdrovieth4-Trjacetjap liaelpne.. 0-CI-hydro, ethyl- , 6imethy1-2.3-di4Ydro...wramo2,4sdions)4, Carbonyl compounds are in general readily reduced to Macon.. dary alcohols t agents such as aluminium isopropoXide, lithium aluminium hydride and potassium torohydride. Although aluninium isopropoxide Se said to be without action oneness (Bickinbottoa, 1937), attempts ere madeto reduce delNydroii, acetic acid W this means.. Thus, the pyrone wistreated:with the alkoxide in boiling toluene and then- nylons as solvents (Wilds, 1947), lb reduction occurred since debydrosoetie acid was quails• titatively recovered at the end of each experiment. When dehydroacetic acid vas treated with aluminium lithium hydride or potassium borohydridwin ether and 5% NaOH, Ivelxwe' timely, some reaction must have occurred since only 5C4C% of the original-pyrons wee'recoverable froM the reaction mixtures in addition to nOromacidicresinous materials. Similar.resulte ,were obtained at OP and at room temperature. Chromatography of the above reaction mixtures; gave only one spot which gave a red colour with ferric chloride, corresponding to dohydroacetic acid. Attempts mere now made to synthesise 3,414YdrOA0.0421- triacetic acid lactone by treating the Grignard reagent of 3-bromo.strlacetie avid lactone with acetaldehyde. Sincie the 33.

solubility of bromide is very low in ether, boiling anhydrous tetrahydrofuran (b.p. 65°) was used as solvent. Mo reaction appeared to occur since the bromide was quantitatively recovered at the end of a number of experiments. These negative results are to be expected since Campbell (1959) found halogen atoms attached to the 4.9yrone nucleus to be inert towards most reagents. Previously, Collie (18911) had shown that the action of alkali on 3..bromowtriacetic acid lactone yielded tromoacetone

rather than 3,.khydromp.triacetio acid lactone, 3-Glyo911044rtacetio_acila Lawton. (3-glycolloy14.cethyl. 2,3dihydrompyran0!2,40.dione). When triacetic acid lactone is treated with acetyl chloride in the presence of concentrated sulphuric acid (Miyaki and laanagiehi 1955) or aluminium trichloride and nitrobenaens -(Boltse and Heidonbleuth, 1958) dehydroacetic acid is forded. In the present work these acetylations wererepeated and yields of 23% and 42% respectively, were obtained. When attempts were / made to prepare 3-(2-chloro)ethyl-triacetic acid lactone by using cbloroacetyl chloride as acylating agent, little reaction seemed to occur since the lactone was recovered in almost quan- titative yields from the reaction mixtures. Furthermore, alkali treatment of portions of the reaction mixtures followed by midis. fioation and chromatography, showed the absence of 3suglycolloyli. triacetic acid lactone. 34.

5-ardromv-DehvOrmicetic Acid. (3.adety14.5.hydroxy..606methy1. 2#3.dihydrow.Pyran-2,4-dione)* Certain pyrones have been reported to be oxidised tor.' Fenton's reagent to give hydroxylated.products. Thus, 41..pyrone gave pyromeoonic acid (3..hydroxr-iopyrona) (ghavalieri, 1947)1 2#6.dimet4144..pyrone-3-hydrorr.2,641nuithyl-4-pyrone (Tickle -and Collie, 1902;(tmealieri, 1947) and meconic acid (3-4vamoriiL 4*pyrone.206.rdicarboxylic acid) 5.4virdraggraseacrda acid (Tickle and Collie, 1902) • Attempts at preparing - 5.hydroxyv-dehydreacetie acid l the action of Penton's reagent Need* Smith and 1958) on dehydroacetic acid were unsuccessful. . Repeated attempts at hydroxylating dehydroacetic acid by. Wenfriendis. model hydroxylatintsystem (Udenfriend. Clark. and Axelrod, 1954) proved to be uneuncessful, sines on3y.dehydro., acetic acid was detected by chromatography of the reaction mixtures. The Structures et certain Lon nee

DEHYDROACETIC ACID TRIACETIC ACID LACTONE 0

III. IV.

HYDROXY-DFINDROACETIC ACID IMINO-DEHYDROACETIC ACID

NH CH CH H2OH 2 3

V. VI. IMINO-HYDROXY-DEHYDROACETIC ACID DESOXY-DEHYDROACETIC ACID

OOH

C:3 VII. VIII. TRIACETIC ACID LACTONE-3-CARBOXYLIC ACID 3-(I HYDROXI)ETHYL-TRIACETIC ACID LACTONE 0 No s / 1 iNss to c 143

CHs

X. 5-11IDROXI-DEHDROACETIC ACID 3-ACETIL-6.IIIDROrt METHYL-2,3-DIRIDROPYRA14 2, 4.41/01E 0 0 C001-1

C143 C Ws C n IJI . 2 84-DIMETIIY1,4-FIRONE 2,6-1414ETIMIr4.-PIRO1S-3-GARBOXILIC ACID. CHAPTER 3 METHODS

The metabolism of dshydroacetic acid, WroArdshydroacetio acid, triacetic acid lactone and iminodehydroaoatic acid have been investigated in the intact rat and rabbit. The mellow* of certain urinary metabolite* were shown by colour chromatography and isolation in the case of inactive feeds and by auto.. radiography and isotope dilution experiments where animals were dosmilvith 140.conpounde, 1400 2 in the expired air WAX estimated as barium carbonate; urines, homogenised faeces and tissues ware counted directly on an end window 13-14 counter. MOtabolies chamber So as to make the collection of respiratory 002 and volatile metabolites possible, dosed animals were placed in assts.. holism chamber (Parke, 19,61 also Oesener, 1958). Air, drawn through the tank by a water suction pump, swept the expired air through an absorption train consisting of the followings a condenser and two glees tubes (30 x 2 cm.) each containing about 40 gn. anhydrone (magnesium perchlorate) to remove the moisture from the circulating air; a test tube containing 15 ml ethanol immersed in a drikold-methanol bath (-70') to condense any volatile oomponents of the expired air; two 38.

Drachma bottles each containing about 150 ml. 2.5N %OH and

fitted with a sintered glass dispenser to absorb respiratory

0021 and finally aftaechoel bottle containing 100 ml. distilled water to prevent loss of alkali by spraying from the previous bottles, It was found that a condenser placed in the absorption

train immediately before the &airdrome tubes removed most of

the moisture from the circulating air* The drying agent was placed in tubes rather than Dreasel bottles since, in the latter, it often formed lumps around the exit tube which on some occasions blocked the passage of air through the absorption train. The animals were given food and water without restriction

(see Appendix III).

Est nation Respiratory 241302 was estimated as described by Calvin, Heidelbergar, Reid, Tolbert and Iankwioh (1910). Thus, 10 ml. each of WE ammonium chloride and 0.641 barium chloride were added to 10 ml. of the Na0HPRaNC03 solution. The mixture was gently boiled for 2•..3 min, and then poured into a tared Gooch crucible of pore size A0-50 microns. The slurry was left in the crucible for about 10 min. before applying auction. This procedure ensured rapid filtration* The precipitated carbonate 39.

was washed with hot water, dried at 110.420° to constant weight and its activity determined tor counting under an end window G-Mcoonter. Chromatoeranhic methods (See Tabbo 6 And 7)

jeotonediXutionexperipents pahydroecetto sop) ( )4 Carrier dehydroacetic acid (0.6-0.9 g. in 10 al. saturated NaH003 solution) was added to a portion of the urine (2-20 ml.) and the solution acidified with conc. H01 to repreeipitate the free avid. This was oollected ter filtration and crystallized from ethanol (charcoal). The crude pyrone was purified by reorystallization from ethanol and carbon tetrachloride to constant specific activity. The acid (a.p. and mixed m.p. 109°) was dissolved in ethanol and converted to the e-phenylhydrawne by the addition of one equivalent of phenylhydraaine in 20% acetic said and keeping the mixture at 80-90° for 40 min. The crude derivative was recrystallized from ethanol to constant specific activity (14p4; admixed m.p. 2D3°). Dehydropoetic acid (total). Carrier dehydroacetio acid(1-1.2 g.) was added to a portion of the urine (2.20 ml.) which was then refluxed with an equal volume of conc. HCLEthanol (0.5 ml.) was added to prevent removal of the steam volatile wone into the condenser. The hydrolysed solution was then continuously TABLE 6 PAPER cmomAToaRAPRT OP SOME PIRONES

0 1 A B C D E Dehydroacetic acid 0.88 0,98 0.98 0.47 0.98 Trimetic acid laotone 0.46 0.14 0.32 0,;39 0.96 Rydroxy dehydroacetic acid 0,75 0.63 0.86 0.29 0.96 Deeoq debpiroacetic acid 0.88 0.15 0,83 0.49 0.96 Imino dehydroacetic acid 0.20 0.54 0,55 0.87 0.91 Ian° hydroxy-dehiroacetic acid 0.05 0.02 0.02 0.79 0.82 2,6 - Dimst41-4-Twrons Ham 0,078 0.74 . 0.83 0.93 2,6 - Dimethy1.4-pyrone 5-carboxylic acid :1):62 0.83 0.95 0.21 0.91 NetaboliteuX" 0.03 0.12 0,15 0.02 0.65 Urea 0.00 0.00 0.00 0.43 0.54 t i

R values are for descending chromatography on Whatman No. 1 paper •in As diisopropyl ether saturated with water (Parket 1960)1 Bs benzene - acetic acid - water (11122 by vol.); Os benzene - methylethylketone r• 22$ aq. formic acid (91111 by vol) (Reio,' 1956). Aa n butanol 56% NH3 - water (4023, by vol.); Ea n butanol- acetic acid - water (43112, by vol.). Table 7. Colour reactiqns of some wrones

119.13 BFBB )01:1011 Dewar wacetic acid reil..oeeme pale pale lilac brown violet Triaoetio acid lantana real violet violet brown Hydrogy- red..oange pale pale lilac brown dehydroacetio acid (green") violet Dosonr- red. violet violet brown dehydroaoetio acid Imino vale's pale pale lilac brown dehydroacetic acid .1 (yellow") violet Imino-hydrozp. yellow pale pale lilao brown dehydroaoetio aoM (yellows) violet none none UMW brown 4opyrone 7:ociet:le violet brown PYrone3 carboxylic' purple acid Metabolite"):" violet .ired violet violet brown (reds) Urea none none

uorescerce wader ult r olet light uenching of background uorescence under ultraviolet light.

The above pyros were ted, OA paper cahiomatograms by the following spray reagents; DAB: wily p-dimethYlaminobellsaldeknie containing 044 piperidine. After sprayinwith thisent,'the chromatograms were heated at 1100 for 1. miss. "/w aq. Fe013. FOBB M/VBrentamine Fast Blues B salt followed by saturated aq. HaD303. Kma04.: 2;4 Afv:MUn04 followed by washing with tap water. Urea was detected as a blue spot by spraying the chromatograms with a (n/v) eq. suspension of Jack-bean meal followed by Bromothymol blue (2cD.H. Indicator Solution). 42.

extraoted with ether for /* hrs. and the ethereal extract dried over anhydrous Ns2$04. The solvent was removed and the crystalline residue of dehydroaoetic acid purified as described above. AXdroV, dehxdraPetk9 acid (prep). Biosynthetic carrier (15..60 mg.) was dissolved in a portion of the urine (24) ml.) and the solution adjusted to pH 3.5 with 24i-HC1 and then continuously extracted with ether for 4 hrs. The ethereal extract was dried over anhydrous Na304, evaporated to about 5 mi. and chromatographed on Whatman 34H paper in solvent B. The band at R7 0.5-0.6 containing hydro* dehydroacetic said was out out and eluted with methanol. The abate vas filtered and evaporated to dryness, and the crystalline reaidta of hydrow- dehydroacetio acid reorystallised from benzene and water to constant activity (insp. and mixedm.p. 1330). The material was counted as thin samples on planohettes using discs of lens tissue (No. 105, Barcham Green Ltd.). &Aro= dehyOroaceic acid (total). Carrier hydrow dehydroacetic acid (5040 mg.) was dissolved in a portion of the urine (2-20 ml.) and the solution refluxed with an equal volume of cone. H01 and 0.5 ml, ethanol for 10 min. The hydrolysed solution was continuously extracted with ether for 3 hrs., the ethereal extract dried over anhydrous Nae04 and concentrated to about 5 ml. The concentrate was chromatographed on Whatman 314H paper (solvent B) and the hYdreW dehYdroacetic acid cut out, eluted with methanol and purified as described previously, impo-debrdroacetic aei#, Carrier imino-dehydroacetio acid (0,5-0,7 g,) dissolved in 10 ml. methanol Was added to a portion of the urine (2.20 ml.). The solution was filtered . and left in a refrigerator overnight, The needles of imino dehydroaoetic acid which separated were filtered off and repeatedly recrystallised from methanol and ethyl acetate to constant activity (mop, and mixed m.p, 20e) ZriecetAc.aaid laotone. Carrier lactone (0.6.0,8 g,) wan added to a portion of the urine (2,2D ml,) and dissolved t,r heating, The solution was adjusted to pH 3.5 with 2 lin/ treated with charms' and rapidly filtered hot. The filtrate deposited long needlowof the lactone.on sloWcooling; these were removed by filtration and repeatedly' recrystallized from water and then ethylmethyl ketone to constant aotivity (114,p. and mixed 14p* 1$8489q), The purified lactons was converted into its 3-bromo derivative according to Collie (18914 and reorystailised from ethanol to constant activity (a.p. 212-214% dectomp.),, Triacetic said leptons was estimated in !teems tr the following method: a solution of the lactone (0.4-0.6 g.) in portion the saturated NOM3 solution (2D ml.) was added a fasoes (1.5 g.). The mixture was homogenized, kept at 60-70° for 10 min. and then filtered through byflo super-eel. The filtrate was adjusted to pH 3-3.5 by the addition of 24[HC1 and continuously extracted with chloroform for 12 hrs.. The boiling solvent was changed every 2 hrs. to minimize decomposition of the lactone. The combined ohloroform extracts were dried over anhydrous Mg804 and concentrated to 1/4 volume under reduced.. pressure' On cooling, crystals of triacetia acid lactone came down; these were collected and purified as described above. , sampAr dehydroaoetie acid. Duos:, dehydroacetio acid (0.5-0.7 g.) was dissolved in a portion of the urine (240 ml.) by beating and filtered hot after treatment with charcoal. On cooling, crystals of desoxy debydroacetic acid came down; these were filtered off and recrystallized from ethyl acetate and water until free from radioactivity (m.p. and mixedlisp. 1850)4 06-Dimetinr).-4-orrena % Carrier pyrone (0.5-0.7 g.) vas dissolved in a portion of the urine (2-20 m1.) and the solution continuously extracted with ether for 3 bra. The ethereal extract was dried over anhydrous Na2SO4 and evaporated to give a crystalline residue. This was repeatedly recrystallized from ethyl acetate and finally sublimed under reduced pressure (0.1 mm. Hg and 80°); the purified dimethylpyrone (m.p, and mixed mop. 133°) showed no radioactivity . 45.

2.6-Dimatkvl-Appyrone...3.carboxylic acid. The acid (0.6 g.) dissolved in 10 ml. N-N12003 solution was added to a portion of the urine (240 ml.). The mixture vas acidified (pH 1) with 2N-H01 and the precipitated dimethyl wone carboxylic acid filtered oft and crystallized from water (charcoal). The acid was recrystallized from water and benzene-light petroleum (b.p. 60.80?) until free from radioactivity (m.p. and mixed mop. 990)• 9gdalas. 0-plotiffixppgrcdaplIA °rano' (0.7 g.) was dissolved in a port on of the urine (2-.2) m14 by warming and the solution acidified to ph 1 with 2N-H01= treated with charcoal and filtered hot. The filtrate was concentrated In vacua, to half volume and left in a refrigerator overnight. The crude crystals of arcinol which separated were filtered off and recrystallized from water and chloroform until free from activity (m.p, and mixed m.p. 107°). Melanie acid. The acrid (0,5.0.7 g.) was dissolved in a portion of the urine (2.20 ml.), the solution adjusted to pH 1 with 2N HC1 and continuously extracted with ether for 8 bre, Crystals of malonie acid separated from the ethereal solution on (tooling) lndewere filtered off and reorystallized from ether and then benzene-light petroleum (b.p. 60-804) until free from activity (10.1P. and mixed m.p. 136°). Sucoinic acid, Carrier suocinic aoid (0.3-0,6 g,) was added to a portion of the urine (2-20 ml.) and the mixture adjusted to pH 1 by the addition of 2N' H01. The mixture was ether extracted for 10 hrs. and the solvent removed under reduced pressure. The crystalline residue was recrystallized from water till free from activity (m.p. and mixed m.p. 184-183°). Oxalic said. Carrier oxalic aoid (0.4-0.6 g.) was added to a portion of the urine (2.20 ml.) which was then made neutral with 2N-ammonium hydroxide. The acid was precipitated as its calcium salt by the addition of a 10% solution of calcium chloride. The preoipitate was collected by filtration, dissolved in 2N-a3D.3 and repreoipitated by neutralization with 211-ammonium hydroxide. The calcium oxalate thus obtained was found to entrain no radio- eetivity. ant Carrier urea (.4- .6 g.) was added to a portion of the urine (2-20 ml.), The solution was filtered (charcoal) and conc. nitric acid added to the ice cold filtrate till no further precipitation occurred The urea nitrate was filtered off and recrystallized from ethanol till constant activity (m.p. and mixed m.p,152°, decomp.). Acetone. Acetone (0.4-0.6 pg.) was added to 2-20 ml. of the urine. 2sirdinitrophenylhydrasine (as an 1% ethanolic solution containing 1% cone, H01) was added till the precipitation was 47.

complete. The precipitate was collected by filtration and repeatedly recrystallized from aqueooithanol (la) till free from activity (m.p. and mixed m.p. 128°). Acetic sad. Carrier sodium acetate (0.3-0.4 gm.) Was added to a portion of the urine (2.20 mi.). The mixture was acidified with 2N-HC1 (to -pH 1.14) and then sutdected to continuous ether extraction (4 hrs.). The solvent was removed on a hot water bath, and the tarry residue dissolved in about 2 ml. water, made just alkaline to methyl orange (pH 4) and then acidified with a few drops of 41-Hol. 500 mg. bengyl idothiurea diesolved in 5 ml. Water was .added and the preoipitate formed after leaving in ice/water collected by filtration. The thiouronium salt was crystallized fro ethanol (charcoal). The Substance was then recrystallized from'ethanol till free from activity (m.p. and mixed m.p. 1.75!)• CHAPTER

THE METABOLISM OP DEHIMAGETIC ACID AND SOME MATED COMPOUNDS IN THE EMIT AND RAT

(a) ChromatosNapbio Investigation* of the Brines 4f anruifia asigdpiatt4re4 qertaioi Prone'

Dohydrcaoet le acid Chromatograms prepared tram the untreated urines of rabbits and rats fed on dehydroacetio acid (DHA) revealed the presents, of DHA itself, hydroxydehydroaoetio acid (05.0HA), iminodehydroacetio acid (imino..DHA), irainobydrovdebydroaoetio acid (iminow0H-DHA), triacetic acid laotons (TAL) and an u.nknovn metabolite "X" by colour reactions (see Tables 6 & 7). In these experiments solvents A, B,CandDwore used. It was noticed that the relative amounts of metabolites depended on the feed level: at a low level (60 mg./kg.) DM end its hydruxy derivative were prawn* in small amounts, whereas at a level of i.94. mg./kg., these two pyronss appeared to account for much more of the dose. These preliminary findings were substantiated by the estimation of the metabolite of DHA in the urines of rabbits administered the radioactive compound by autoradiography and strip counting (solvents A, B and Cs see Table 8). Autoradiograms prepared from the untreated urines of rats fed on tL40 I.DHA revealed a rather siriAlAr metabolic pattern. TABLE 8. THE URINARY METABOLITES OF [14*DEHXDROACETIC ACID IN TRE RABBIT ESTIMATED ALAUTORADINRAPHI AND SBI Comm

Does Level Metabolite 60 wg./kg. 494 mg./kg. (Rabbit No. 5) (Rabbit !b.

Dehydroacetic acid 6 7 Rydrowfdehydroacetic 14 7 void Iminodehydroacetic 5 16 acid Iminohydrovdebydrp.. 5 acetic acid Triacetio acid lactone 5 Metabolite "X" 22 2 Metabolite! * 13 3

Urea 1 .

Metabolite failed to give any colours with the detecting agents.

gat; , 049 Appydroacetia mid in the lipian. A male human subject (67kgm.) was fed 141 ap.DHA di000lved in 4, ml. saturated NaHCO3. (2.1 mg./kg. or 0.013 meerale/kg). The urine passed ]3. bra. later (340 ml,) was collected, adjusted to'pli 2.5-.3 with 254101 and 49.

continuously extracted with ether for O hours. The ethereal extract thus obtained was dried over anhydrous M004, and then reduced to dryness on a hot water bath. The residue was dissolved in 2.11. methanol and portions (0.1 ml.) chromatographed on Whatman Vb. 1 paper in solvents B and D. • The usual detecting agents revealed.the presence of DHA and 0H-DHA in about equal mounts (5-10% of dose) and also of TAL and metabolite (24% and 20%. respectively). late of dehTdroacetie acid in thelyan. 105 mg, DHA dissolved in 10 ml. saturated 110003 was administered by means of a crop tube to a hen of weight 1.5 kg n. (70 mg./kg. or 0.41 m.-mole/kg.). The 24 hour excreta (urine and faeces) were collected and adjusted to. 01. 2.54' witk25..H01..The mixture was kept at 60.100 for 5 min. and then filtered through Hyflo Super .Gel. The clear filtrate was continuously extracted with ether fer'6 hours and the ethereal extract thus obtained dried over anhydrous Mg604.and then reduced to e. tar on a hot water bath. This was taken up in 2 ml. methanol and portions (0.1 ml.) chromatograOhed:on Whaiman.No. 1 paper in solvent', B and D. The usual detecting agents revealed that WA and 05.DHA were present in aboutequal amounts (10-20 of dose) and that- TAL and metabolite "X" were also present (1..2% and 20-30% respectively). Tniacotio acid_ Mot9c4al Autoradiograms (solvents B and D) prepared from the untreated 50.

urine of a rat administered 364.3 mg./kg. [1%14Alerevealed that the lactone and urea were the only radioactive substances present. The urine of a rabbit administered 53.6 mg./kg. of [2403J-TAL when sidtilarly treated obeyed the lactone to be present at atOut 15-20% of the dose; in addition, radioactivity corresponding to about 10% remained at the origin. This material. failed to give am/ colours with the detecting agents. Imino-dehydroacetikagAi When chrematographing a portion of the untreated urine of a rat administered 229.6 mg./kg. 0404j-imino-DIJA acid the absence of imino-011-DHA acid was conspicuous (Solvents B and D). Iiiino-DHA acid was the only compound revealed by the usual colour reagents, autoradiogram showed the compound to be present at about 20% of the dose level. An unknown metabolite of values 0.15 and 0.05 in solvents B and D respectively was present at 40% of the dose level; triacetic acid lactone was just deter" table by this method. (4; 1%). Sardroxrdebvdroacietio snit Colour chroiatograme (solvents B and D) prepared trom the 48 hoUr untreated urine of a rat (0.145 gm.) administered OSZHA acid orally (22.3 mg.; 154 mg./kg.. or 0.84m..40,10/kg.) showed the original-Pyrene to be present (10%) in addition to TAL (10%) andAnstabolit4OW 120%) 72 hrs after-dosage the animal was 51.

killed by stunning and blood (2 ml.), the liver and kidneys removed and homogenized in a Potter.Elvhjan homogeniser. The mixture was adjusted to p11 2.5-3 by the addition of 2frH01 and then continuously ether extracted for 3 bra. The ethereal extract was dried over anhydrous Mg804. and reduced to dryness. The residue was taken up in 1 ml. methanol and portions (0.2 ml.) chromatographed (solvents B and D). This revealed the presence of OB4HA. (1..2% of dosage in total blood.112 ml. 7 % of b047 weight) liver and kidneys). (b) The IaoXation or MOtiebolite Pam the Urines of Two _Rabbits administered Dehvdrovetic Agit A total of 5 g. DHA (800 mg./4.) as a 12.5% solution in saturated aquas NaH003 was administered by stomach tube to two female chinchilla rabbits and their urines collected for three days and stored under toluene (2 ml.) at 100. The bulked urine (960 ml., pH 8.5) was filtered through Hyflosuper-Gel, adjusted to pH 3.5-4 with 21441C1 and continuously extracted with ether for 8 hours. The ether in the reservoir vas changed every 0.5 hrs. to minimise decomposition of the pyrones. The combined ether extracts were dried over anhydrous Ha2S 4 (charcoal), filtered and evaporated in vaouo to give a dark yellow semi-crystalline residue (5,1 g., m.p. A5-50°). This was dissolved in a mixture of benzene (p ml.) and methanol (10 ml.). The solution separated into two phases, 52.

The upper phase was concentrated to about half its volume and left overnight when long needles of Prdroxydebydroacetio acid separated (350 mg., .7%, of. dose). These were removed by filtration and recrystallized !roe benzene and then water to give the pure compound of m.p. 133.5°. The benzene mother liquors of the upper phase were evapotated on a hot water bath and the tarry residue dissolved in hot ethailol and filtered (charcoal). On cooling the filtrate, crystals deposited which were filtered off (605 mg., equivalent to 12.1% of dose) and recrystallized from ethanol and then C01 give debripoacetiq 4 to, m.p. and mixed m.p. 109°. This was confirmed by prepating the l'avbenylhydrasone (Perkin, 1887)1 m.p. and mixed m.p. 202..3°.

The lower phase was concentrated in vacuo to about 2 ml. and then subjected to large scale paper chtomatographron Whatman 3MM paper in solvent B. The bands at EF 0.11-046 (metabolite "X") and 0.60-0.70 (OH-DHA) were cut out and eluted with methanol. The eluate of the first of those bands was concentrated pi vaouo to 3 ml., an equal amount of etIyl acetate added and the solution left overnight at -10° when buff needles separated. These were filtered off (18.2 mg. or 0.0 of the dose) and recrystallized from methanol to give colourless needles, m.p. 182° (decomp.) chromatographically pure in Solvents Al B, C, D and E. The eluate 53.

of the second band was evaporated to dryness on a water bath and the crystalline residue (405 mg., equivalent to 8.1% of dose) recrystallized from benzene and then water to give a second crop of hydrozydehydroacetic acid, m.p. 133.5° (Total yield of hydroxydehydroacetic acid, 15.1% of doe.). (c) Ideptificulppn and Pronexties of Mydroxydehrdroacetp acid As described above, a compound of m.p. 13305° was isolated from the pooled urines of two rabbits dosed on dehydroacetic acid.

(Found, C, 52.01; B4 4.45; C0805 require. C, 52•201 H, 4438). The metabolite was very soluble in methanol, ethanol, acetone and ethyl acetate; lees so in water, benzene and chloroform, and only sparingly soluble in light petroleum (b.p. 60.40°). It reacted with the colour reagents and had Pit values as described in Tables 6 & 7. The presence of an hydroxyl group was shown by a positive vanadium oxine test"(Feigl, 1956) and also by infra red spectros- copy. Theie preliminary findings suggested that the compound might be.an Wray derivative of debydroacetic acid. Rgydroxy dehrdroacetio acids has fOur possible structures,- 54.

COCH2OH

zu IV.

111UX011102r0111411

chromophato

K bands due to conjugated system (220-250 ma). MI bands due to the displacement of the band of the carboxyl group from 270 mu to 300-320 me. 55.

The fact that BHA and °H-DHA have very similar ultraviolet absorption properties (see Appendix I) suggests that an additional chromophore has not been introduced. This argument eliminates IV since an hydroxyl group in the 5 position would result in a marked battlychromic shift of the a-band of BHA (308 towards the 270 so region. Since the new compound is acidic - indeed it has a pita of 5.0 thus being more acidic than DHA (pKa 5.3) - and strong1T enolio. structure II must be eliminated. New. the compound was found to be oxidised by Fehlingts solution and Benedictwe reagent after boiling for 2-3 min. and to give a silver mirror with Tollenos reagent on gentle heating. These reducing properties are not shown ter PHA or TAL even after prolonged boiling. This strongly suggests that structure I should be assigned to hydrovdehydroacetic acid. Final proof of the-structure of the compound was given on its conversion, to TAL. aga. 113 mg. 0,11..DHA (chromatographically free from DHA and of 'up, 133.5°) was treated with 0.3 ml, 90% 8204 (v/v) at 125-10 for 5 min. The dark-brown mixture was cooled and powdered ice added to make a final volume of about 2 ml. and then continuously extracted with ether until it failed to give a red colour with Fe013 (12 hrs.). The ether in the reservoir was changed every hour to minimise decomposition of the pyrone. The pooled extracts were dried over anhydrous M8SO4 and concern.. trated to give a tarry residue. This was dissolved in 1 ml. methanol and the solution thus obtained subjected to chromate)... le graphic separation on Whatman 31414 paper -ark solvent B. The paper 56.

vas overrun for 15 hrs. and then dried at 60..70° for 4 bra. The tend corresponding to TAL was cut out and the pyrone eluted with methanol. The Sluate (about 2 ml.) was filtered and the solvent removed on a hot water bath in a stream of N2. The tarry residue (10.1 mg.) was taken up in 1 ml. water,and filtered warm in the presence of charcoal. The filtrate was concentrated in vacua (over KOH) to about half its volume and then placed in a refrigerator for 3-4 days (5-6°). Needles of triacetip acicj IpOtone (m.p. and mixed m.p. 188°) came down (4.2 mg., 5./i on 05.0HA),

Tri ee c acid lacto bromide was prepared according to Collie (1891): 3.6 mg. of. the material obtained above was dissolved in 0.6 mlo glacial acetic acid, The mixture was cooled in ice/water and bromine added till the solution was permanently yellow-orange. The precipitate Which slowly formed was filtered off and crystallised from ethanol to give 3.54 mg, (61%) trkatetio acid Inctonit-3ibromida (m.p. and mixed m.p. 21244% decomposing).

The presence of. TAL in the reaction mixture of the ndeglyeolloylation" of OH-DHA was also hewn . chromatography in solvent D* The wone gave the usual:colour reactions on paper. Residual OH-PHA could not be detected by this method in the reaction mixtures. A second compound remaining , at the origin 57.

when solvents B and D were used, gave the usual colour reactions and appeared to be a major product of the degradation. This was not identified. Derivatives of hvdroxy dehydroacetic eat loinolhArezrdehyOreseetic acid. 92 mg. OB.DHA were (d = 0.92) giving a pale biege solution dissolved in 1 ml. aq. NH3 and on leaving overnight a white precipitate came down. This was collected and crystallized from methanol and then from ethylacetate to give Amino-40m dehydroacetic ac 4, (Found, Co 52.441 Hp 4.943 Ns 7.64 C8N04 requires C, 52.79; H, 4.68; N, 7.62). The colourless mierocryetalline compound (needles) had m.p. 224"5° and was obtained in a yield of 50% (42 mg.). NonoseMicarbazonet 106 mg. 011..DBA were dissolved in 15 ml. water containing 1 ml. ethanol. To this was added a solution of aemicarbaaide (53 mg.) and crystalline sodium acetate (69 mg4 in 0.5 ml. water. The mixture was stirred and left overnight when a white crystalline precipitate came down. This was collected and recrystallized twice from methanol to give lurdrozy-dehydroacetic acid monosimicarbasone AFoundo Co 44.5; 11! 4.9; N, 17.8; C9H211505 requires Co 44.8; H, 4.6; Bo 17.4). The derivative (needles) had m.p. 1954° (decomp.) and was obtained in a yield of 52% (81 mg.). 58.

(d) Some Properties of Metabolite 'IX"

Metabolite "X" has itr values and colour reactions on paper as shown in Tables 6 &7. Like DHA and TAL but unlike O&M, the compound did not reduce Fehling's solution, Benedict's or Vollen's reagents. On titration ageinstOtO2Ria0H, an apparent Ai of 4,9 and equivalent weight of 237 were obtained. The metabolite Jo, therefore, monobasic and of the same order of acidity as TAL (pK 4.9). The metabolite shows an ultraviolet absorption curve consistent with a pyran-24,4*dione structure that is, it shows a marked bath/chromic shift on passing from acidic to alkaline solution. Whilst'the K.-band of the compound occupies the same position of the spectrum as those of DHA and OH-DHA, the R-band shows a, hypmmhromio shift of about 15 mp. These findings suggest that a new chromophore has not been introduced but that some modification to the aumcchrome has occurred instead.

Tables 9 and ID show the fate of [14C4J4HA and [14 J-TAL in the rabbit and rat when given orally and Tablell summarises the results obtained when [1404)-imino-DHA was administered te, the same route to the rat. When the rate: is injectedsubcuto. aneously with 14C47.DHA (Tablet?.), results are obtained which 59.

are, twofer as can, be seen, identical with those gained when the compound VAS administered by stomach tube (figs 2 & j). The rates of excretion of 14002 in the expired air and total radioactivity in the urines of two rabbits fed on [14C4).MHA and [403,1-TAL, respectively, are compared in figs. 4 & 5. From the above, it is evident that [403J.TAL is much more completely and rapidly metabolised to 4002 (45-50%) in the rabbit and rat than (4041411A. Also, the tissues of rabbits of thedose 2, 3, and 5 were found to entrain 11.5%, 10% and g%; respectively seven days after dosage. [404,14mino-DHA was found to give rise to very little respiratory 14002-in the rat (2.3%); here the venal route appears to be more important in eliminating radioactivity. Table 11 shows that 60% of the activity fed is excreted in two dam when [14e4J-DHA is fed less than 20% of the activity is eliminated in this time. The rather high total radioactivities .(18%) found in the faeces of rats dosed on [C4).4iHA is explained by the elimination of radioactivity the bile. The total radioactivity of the faeces of rats given 1494raorally can only be explained by incomplete absorption of the lactons since [40 J-.TAL does not appear to be excreted nfi the bile. 60.

c-• 14 TABLE 9. THE FATE OF ..DIARMROACETIO ACID X4111) RAT

Rabbit Hat 00 10.1 (3.2) 11.9 (2.6) 2 in expired. air Dehydroacatic acid (total) 5.5 (1.8) 4.7 (1.4) Rydrovdehydroacetio acid (total) 21.3 (0.0) • 8.0 (0.3) Triacetio acid lactons 9.3 (2.6) 1.4 (0.4) Total metabolites in urine 33.1 (2.6) 24.1 (3.6) Total radioaotiVity in urine 66.3 (11.8) 22.8 (6.8) Total radioactivity in faeces 3.8 (2.1) 17.8 (5.1) Total radioaottvity in tissues 12.4 (3.7) 14.7 (84) TotalTadloactivity accounted for 92 .(7) 77 (5)

(The figures are arithmetic:Immo of those given in Tables 21 and 22 Rabbit 4. and Rat I have beenexauded; the former because of its high dosage level, the latter as being abnormal. The standard deviation is given in parentheses).

T 10.

b t Ra t 002 in expired air . 54.1 (6.6) 46.1 (0.3) Triacetio acid Inatome 16.4 (6.1) 19.7 (4.8) Total radioactivity in urine 29•4. (6.2) 20.2 (4.8) Total radioactivity in faeces 3.8'.1 (1.5) 6.7 (2.3) Total radioactivity in tieSUea' 13.7 (.6.4) 1206 (6.5) Total radioactivity accounted for 101 -(2) 85 (3)

(The figures are means of those given, in Tablet 23 and 24. The standard deviation is given in parentheses) 61.

Tablell The fate of "Ct Imino-de roaoetio acid in the rat (adminieter orally as a suspension in water oontaininK 10 mg. bile mate)

Ahimal 1 2 Body weight (kg.) 0.25 .0.15 Dose level (mg./kg.) 229.6 237.1 (no-mole/Kg.) 1.38 1.42 Does of radioactivity (p) 1.8 1..09 Duration of experiment (days) 2 3 percentage of dose CO2 in expired air 2.1 3.4 Debydroacetic acid (free) Os Os Imino dehydroacetic said 14.7. 15.1 Hydroxy dehydroacetio gold (total) 01 el Triaoetio acid lactone 0.4 0.9 Urea 0.1 0.2 Total metabolites in urine 15.2. 16.2 Total radioactivity in urine 59.4 60.9 Total radioactivity in faeces 13.9 19.1 Total radioactivity in tissues 1>3 1.2 Total radioaotivity accounted for 78 85

1> 0,03% le* 31. 0.04% 62.

Table 32 The fate of 140 - Dehydroacetio acid in, the ro (a nistered b, sob- cutaneous in4eotion

Animal 2 Body weight (kg.) 0.27 0.45 Does level (mg./ g.) 63.8 99.3 (mrmole/kg.) 0.30 0.59 Dose of radioactivity (m-o) 4.93 7.90 Duration of experiment (days) 3 use CO 2in expired air 104 5.1 Debydroacetio aoid (total) 2.3 . Hydroxydehydroacetio acid (total) 10.1, 4.4 Triacetio acid lactone 007 . •Urea - 0.3 .... Total bolit in Urine 13.4 Total radioactivity in urine 33.0 : 17.1 Dehydroacetio acid (total) 2.2 1.6 Hydroxydohydroacetio acid (total) 7.5: 3.5 Triaoctio acid laotone : 0.011 0.0m Total metabolites in faeces 9.7 5.177 Total radioactivity in faeces 13.4 8.5 Total radioactivity in gut contents 5.3 Total raaioaotivity in. tissues 15 39 Total radioactivity accounted for 72 75

1 j> 0.03A t includes gut contents In rat 2. DHA. OH-DHA and TAL were estimated in the gut contents only. 7c+ CO 410 40 Bo. 0 60 T om= "ersAr tzv3 ).7.115* OlczetIrt)

1./7715 lieftWe MArb Co2. a- .2, 5-xcRATIWV ®F' 7t2TITI. 1 t ;11)10 19trIk 7 "y

X/1, 774F E pnel () y Rxri- wacti 1,149 REcarzyt,a7!) ne-

OF {144 -7. 410 60 AP A1V L)056" eatIRS)

/47/6-. 3 R E•77 W of 7V179A. 47R3/0 i3entefrY rm' ?WE' unit,E 641) AI/51) Co

rAle EArPPIE;) /WA' N 871 Fit Rivr Aviv/chi N RaciGivd 63-Swo- op f)/R11(6- g"./ Svtgevigivii&-ovS 2-AvIR:cmcw 7 •••••••••

5 Ty mE MollI g) P/At

7077L. Vgioser 12 hVi ofiwri )07'Y

/46f1.1/4, rtar —0— Ak.11Mg ••• pro/4491R 53. b /011/.ta _ _ ..... 7brifit, vgififr f7Yea7 ._.44 Rpoog...ry Ty egilid-y 4

1.1110.010101•••••••1171.4•••••••••••••••••=•.orime..• le 10 30 N0 TIME RFrirl? DoSI 1/44040 G0 /0 1Oa

Ff‘4 C041,4.414:7,-- , of THE Svc.fra-new Mr44 R911096ninry 74, 77 Vtirna R/V) "CO -r/V E R g) Pi R CF Two AZ RaeirS Gira 4, Et I - g ,r7,9 J -7PL R ) 1 t'Afoir.f4.1 tcr 7/Alf C AFAWAir ,isZci9coF

giSt. S 7NE fe,p7E 6-,,c4,07/04-' n F 4vCc2. TN 7-rve

OF 71.vc Rivegfis. c 60 dra/46-Eveq-)1.111. (I) 9 /0

/17 E.Sicsc: E1- 7' 5-3' /11 k r1 C3 - r4) 1 40 77- nrie771-4 ler:ArOC 4;(1.06,5r,i)

ff co?Cr idly O F 77/5. 7C37i R/39/0 erit a: 7/ te/7y rfiF v4r,e4-7 or 77 Virzi"' rs-IMOY R A-ChS:1114

/771 e- arc' Ellid- ;,t,.)prio -Pill; 14-6, oftp,i.4-/ The urinary metabolites of [14C4J-DHA in the rabbit and rat are compared in Table 1.1

TABLE 13. ARING THE URINABX MTABOLITES OF I.DEHYDROACETIC.MD IN THE RABBIT AND RAT (The figures are percentages of the total urine radioactivities)

Bat Dehydroacetic acid (total) 8.3 20.6 Hydrovdehydroacetic acid (total) 32.2 35.1 Triacetic acid lactone 14.0 6.1 Total metabolites in urine 54.5 61.8

In keeping with the results obtained by'chromatography (Table 8)1 the method of, isotope dilution' showed that at a high dose level (Rabbit 4, Table 211) DHA and OH-DHA account for more than 50% of the dose, whereas at lower feed levels, the two pyrones account for less than 25% of the total urine radioactivity

(f) dehrdpmptis Acid tOeir .reppoctiva Imino- pocuounls is Rabbit end. E *lino It will be noticed from Tables 21 and 22 that the figures given for "dehydroacetic acid, free" in the urines of rabbits and rata administered [1404).DHA are rather erratic - • 69.

varying from 04% to 64 in the rabbit and from 1.6% to 9.4% in the rat, Because of the facile reaction of DHA.with ammonia to form imino.DHA, the possibility that such a combination might occur in normal rabbit and rat urines was investigated. OH-DHA, because of its close resemblance to DHA, was similarly examined. Um, DHA (4.46 mg. in 0.5 ml. water) and OH- 1A (4.25 mg. in 0.5 ml. water) were added to 4.0 ml. fresh rabbit urine. Similarly, 5.15 mg. DHA and 4.65 mg. Oil..DHA were added to 440 ml. fresh rat urine, After mixing, the solutions were kept at le and portions (0.05 ml.) taken at intervals and chromatographed on Uhatman No.. I paper in solvent A. The positions of the four Prrones on the chromatograms were detected ter sprsying duplicate chromatograms with the p-dimethylaminobensaldehyde reagent, and the compounds were separately eluted with ethanol and their concentrations in the eluates determinsd speotrophotometrioally in a

Mist= SF 500. The presence of the imino compounds was confirmed by chromatography of portions of the urines in solvent B. The results (Fig* 7 and Table 27) show that in normal rabbit and rat urines, both DHA and OH-DHA are readily converted into their corresponding imino derivatives. The above was supported by estimating the total and free DHA and OH*.DHA and also imino-DHA in the urine collected directly from the bladder of a rabbit (tb 6) administered [140 J-DHA and killed leo

rMc c 0 vv. 4s? 0 or (0) ,V/ts ON- ipve (A) 70 7116/4 "mil'- co/VAN,A414,S zy RiP4:6/7 73..

three days after dosage.. Imino-DHA could net be detected by the isotope dilution technique and there was little difference between the free and total pyrones. (g) Tissue Slice Experiments Male rats were killed by stunning and the required tissue (liver or kidney) rapidly removed, cooled on crushed ice and place in ice.- cold medium of pg 7.3 (see Table 15). The weighed tissue was cut into slices by the free-hand method (Elliott,' 1955; Cessna:6f 1958) and placed in 20 ml. medium containing approximately 1073.M substrate ([1404j-DHA, and [140)4AL). The tissue-medium-substrate mixture was incubated in an atmosphere of morgen for 1 hr. at 37° in a metabolic Shaker, any CO2 liberated was entrapped in 2.5N-Na0g and counted as BaCO At the end of the incubation period, 5 ml, 50% trichloressetic acid was added to ensure the liberation of arly

"bound" CO2 and the precipitation of soluble proteins, The latter, with the cell debris, were removed 1y centrifugation (5000 g., 5 min.) and then resuspended in 10% trichloroacotic acid and reeentrifuged. This was done t4ice. The :supernatants were pooled and the total DHA and[0341gA and TAL•present estimated by the usual isotope dilutionmethods. The results obtained are summarized in Tables 1649. Both [140 ]-DHA and [1,403j-TAL gave rise to 14002 in liver slices. The kidney was found to be 3-4 times more effective in 72.

oxidizing [14C3J.TAL than the liver but 1/4C4J4EA was apparently stable in the presence of kidney slices. Both 0H-DHA and TAL were shown to be metabolites of DHA in the liver by isotope dilution; of these only OH.DEA could be detected by colour chromatography in addition to DEA. (Solvents C and E). Metabolite X could just

TABLE 14. compariq2 the producIienk ot 'co by tissue slices inaubated with, 140 subetatqa

(The figures, given to three decimal placee, are percentage 14002/gm. wet weight tisauebour). Lint pdnev gadElla. 1 2 3 1 2 3 [1404] DHA 0.014 0.015 0.095 0.002 0.002 0.001

(1403-1 TAL 0.235 0.261 0.373 0.910

be detected by colour chromatography (11%) as a metabolite of DHA in the liver, but not in the kidney. Neither imam-DHA nor amino- OH -DBA could be detected as metabolites of DHA in liver or kidney slices. [1C31-TAL gave rise to neither DHA nor 0E-D in the presence of liver or kidney slices (isotope dilution and colour chromatography). For this reason the deacetylation of DHA in the liver is probably irreversible. TAL did not give rise to metabolite X (colour chromatograpbT). 73.

TABLE 15, COMPOSITION OF t4ED;134 USED in vitro WORK

(thibreito Burris and Stutter, 1949; Krebs, 1950).

All the constituents were made up in freshly boiled glass distilled water.

MaC1 0.15 0.90% 96.2 parts K Cl 0.154 1.15 4.0 KH2PO4 0.154 2.11 1.0 MO04.71120 0.154 3.82 1.0 Nairn°, 0.154 1.30 3.0 Glucose 0.300 5.40 5.0

3.8 parts Sodium phosphate buffer namely, B422PO4.2H20 0.1M 1.78% 100 parts NaH2P0 420 0.1 1.38 25 74.

TABLE 16. The fate of e"161:12ILL2 Rat liver slices

Experiment no. 1 2 3 Wet weight of tissue (gm.) 2.1 1.9 3.5 mg. substrate ([1404j*DliA) 3.14 3.19 3.55 mg. substrate/gm. tissue 1.05 1.68 1.01 mg. substrate/kgm. tissue 142' 146 151 medium mixture 3 Molarity of substrate (z /0'4) 8.4 8.6 10.6 Activity (pm) 1.61 1.64 1.85

percentage of substrate 1400 2 0.03 0.03 0.2 Dehydroacetio acid (total) 97.2 101.0 74.6 4ydroxydehydroacetic acid (total) 1.2 0.8 2.6 Triaceti© acid leptons 0.5 004 0.9 Total metabolites in supernatant 98.9 102.2 78.1 Total radioactivity in superantant 97.0 96.0 99.0

75.

TABLE 17. IljaiLtedt.21[14041.1XIA in rat kidney slices

Experiment no, 1 .2 3. Wet weight of tissue (gm0) 2.2 2,5 4.4 mg. substrate ([1104]-DHA 3.15 3J7- 3.55 mg, substrate/gm tissue 1.44 1.35 0.81 mg. substrate/kgm, tissue-medium 17 142 150 133 mixture 1 Molarity of substrate (x 10"4) 8.4 9.0 10.6

Activity. Pc 1.62 1.73 1,85

percentage of substrate

34002 0.003 0.004 0.002 Dehydroacetic acid 96.0 99 2' 89.1 4ydroxydehydroacetio acid (to ) 1' 0.01 1,0.008 0,005 Triacetic acid lactone , 1©.002 14.003 C).002 Total metabolites in supernatant 9640 99.2 89.1 Total radioactivity in supernatant 100.0 96.0 98.0 76.

TABLE 18. !be fate o; 2193ITALia rat liver taices

Experiment no. 1 2 Wet weight tissue (gm.) 2.6 3.9 mg. substrate ,((140 j...TAL) 2.19 2.47 mg., substrate/gas tissue 0.84 0.64 mg.: substrate/kgm. tissue- 97 104 medium mixture Molarity of substrate (x 104) 7.6 811 Activity (0) 0.092 0.074 percentage of substrate

002 0.61 1.02 Triacetic acid lactone 90.1 ' 88.1 Total radioactivity in . 3 supernatant 97.2 9346 77.

TABLE 19, The fate of (1.149,31-TAL ip ritt kicInex sum

Experiment no. 1 2 Wet weight tissue (gm.) 0.8 1.0 mg. substrate ([14C3J-TAL 2.44 ?de° mg..substrate/gm. tissue 3.05 2.40 mg. substrate/k: • tissue- ) 116 134 ' medium mixture Molarity of substrate (x 10-4) 9.1 8.9

Activity (00) 0.124 0.900 percentage of substrate 0.46 CO2 0.91 Triacetio acid lactose 85,5 92.4 Total radioactivity in 93.2 101.1 supernatant 78.

(11) be Plasma Bindj.nc og DenvjgroAoOlio 14.914 A well known property of plasma albumin is its ability to combine with anions (Foster, 1960). It.was not surprising, therefore, that Woods et al. (1950) found DHA to be present mainly as a non dialiyaeable form in the plasma of doge previously dosed on the compound. This finding its supported in the present concentration of work by showing that, in Atrop [1404 J-DHA at a A5.5 mg. per 100 ml. of serum combines readily with plasma albumin. am, 0.91 mg. 04 4)4)HA (0.467 pc) was incubated with 2.0 ml. human serum (obtained from the Department of Serology, Wright-Fleming Institute) in a metabolic shaker for 1 hr. at Ye. A portion (0.02 ml.) was withdrawn and subjected to paper electro• phoresis on a strip of Whitman No. 1 paper, 5 cm. wide in 0.05M veronal buffer at p11 8.66. The potential applied across the paper was 340 V (12.2 Vbm ). After 5 hours, the paper wall withdrawn, dried in a current of air at 90400! and then cut in half to provide two strips each 2.5 cm. wide. Strip No. 11, , This was strip counted under an end-window 01441 counter, 3. cm. at a time. ptrip No. 2. This was dipped in a 0.1% solution of bromophenol blue in ethanol saturated with mercuric chloride. The excess dye was removed by rinsing in 0411-acetic acid and then in tap water. 79.

The proteins showed up as blue ribbons (Lederer, 1955). Table 20 shows the migration of certain pyrones compared with plasma albumin. Using the method of paper electrophoresis, therefore, it was possible to separate free DHA from albumin-bound DHA. The results obtained when human serum containing [140.4)..DHAmWs subjected to paper electrophoresis is shown in 8.v.

TABLE 20. Comparing the„mierkitionof pertain =ones w,L ,h tAat of albumin in0,105M.veronal WPM% pli 8.4-0„iiih5t41 3). -14 ;Were 12:2 Vc.P.

DistAnge travelled, Method of from link*? flatecttoq application

Metabolite X * 8.5 cm. p-dimethylamino DHA + 8.0 benzaldetorde OH-DHA + 8.0 reagent. TAL + 8,0 brentamine fast blue Albumin 3.4 bromophenol blue NH'011-DHA •2.1 p-dimethylamino BHA - 2.3 benzaldehyde reagent.

80.

1 34 TABLE 21a. The fate oft- 0q-Dehydroacetic acid in the rabbit ' administered orally 48 a solution in_saturated as• Na 40031

Aniznal. 1 2 3 Body weight (kg.) 3.1 2,0 4.0 Dose level (mgt(kgs) 184 7845 10.7 (m.mole/kg.) 0,11 ' 047 0.06 Dose of radioactivity 9Ao) 31.5 12.6 22.2 Duration of experiment (days) 4. •7 7 percentage of dose

002 in expired air 7,2 7.9 104 Debydroaoetio acid (free) 0.5 0.8 2.5 Dehydroacetic acid (total) . Imino dehydroaoetic acid • . . gydroxy dehydreacetio sold (free) 3.6 Hydros y dehydroacetio acid (total) Triacetio acid lactone 18.5' 12.2 8.8 Urea 0.3 0.3 0.5 Total metabolites in urine 19.3 13.3 15.4 Total radioactivity in urine 73.2 79.7 51.0 Total radioactivity in faeces 2.7 •1.7 17,6 Total radioactivity in tissues 16.5 11.5 10 Total radioaotivity accounted for ' 99 102 89

81.

TableZIkoThe fate- of 1; - Dehydroacetic acid in the rabb t (administered orally as a solution in saturated 84./4=0

Animal 4 5 6 Body weight (c6.) 4.0 3.2 2.9 Dose level (mg./kg.) 494.0 60.0' 59.1 (m-molo/kg.) 2.9 0.36 0.35 Dose of radioaottvity (i,w) 13.3 16.3 13.3 Duration of experiment (days) 10 7 3 percentage of doss CO in expired air 2 9.8 15.4 Dehydroacetic acid (free) 24.0 3.8 4.5 Dehydroacetio acid (total) MI6 6.1 4.6' amino dehydroacetic acid 2.5 0* Rydroxy debydrosostio acid (free ) 13.1 19.2 Hydroxy dohydroacetic acid (total) 21.it 21.5t Triacetio acid lactone 3.3 7.114 54.7: Urea 0,1 . Oat 0.21 Total -metabolites in twine. WA 34.9 31.3 Total radioactivity in urine 70.8 71.3 52.3 Total radioactivity in faeces 4.5 3.1 7.1 Total radioactivity in tissues 8 16 Total radioactivity accounted for 83 92 91

f the total metabolites is the sum of the metabolites marked thus. 0.01% 82.

Table 22a The fate of {1401 - Dehydroacetic acid in the rat 9' (administered oral as a solution in saturated ao.NatiOC)3

Animal 1 21IE 3 Body weight (kg.) 0.4 0.37 0.22 Dose level (Tagijkg.) 63.0 17.6 112.0 (m-mole/kg w) 0.41 Q.11 9.67 Dose of radioactivity (AA0) 14.2. 3.7 13.1 Duration of experiment (days) 4 4 4 .percentage of dose

CO in expired air 25.8 2 7.9 14.3 Dehydroacetic acid (free) 1.6 2.4 9.4 Debydroacatic acid (total) /mina debydroaoetia acid Hydroxy dohydroacitie acid (trim) 13.5 Hydroxy dohydroacetio acid (total) -, Triacetic acid leotone 1.3 13.9 2.1 Ursa 0.6 0.3 0.3 Total metabolites in urine 3.5 16.6 25.3. Total radioactivity in urine 45.2 50.7 39.7 Total radioactivity in faecas 11.2 7.8 13.1 Total radioactivity in tissues 3.5 .5.5 13 Total radioactivity accounted for 86 72 8o

a male animal

83.

Table 22bTha fats of 40 - Dehydroadetio acid in the rat a stored orally as a solution in eaturatedag.NaH0031'

Animal 4 5 6 Body weight .(kg.) 0.46 0.16 0030 Dose level (mg./kg.) 58.P 58.5 60.0 (m-mole/kg.) 0.35 .4.55 0.36 Dose of radioactivity (,Ao) 4.7 4.7 9.2 Duration of experiment (days) 5 '5 percentage of doss C©2 in expired air . 12.6. 19+9. 13.6 Dehydroacetio acid (free) 54 „24 3.5 Dehydroacetio acid (total) '6.1 2.8 50 ammo dehydroacetio acid 1.3 1.2 2,6 Bydroxy dehydroaeetke acid (free) 4.6 3.2 5.2 11.74roxy 4ehydroacetie acrid (total) 90 4.0 9.0 Triadetid acid laotone /4 1.3 1.0 • ' 1.4 Urea r 0.2 0.2 0.2 Total metabolites in urine: 16.6 10.0 154 Total radioactivity in urine 23.3 15.8 29.3 Total radioactivity in faeoes 21.5 19.2 16.3 Total radioactivity in tissuea '21 25 20 Total radioactivity accounted for 78 71 79,

The total metabolites in the urine is the 6= of the metabolites marked thus. $4.

Takeer 23 Pa e of 14 * -Iriacetic acid laotone n t a/e rabbit (adminir. stored orally as an aquas solution

Animal 1 .2 3 Body weight (kg.) 2.8 3.3 3.0 Dose level (mg./kg.) 53.4 49.7. 56.7 (m.colaikg.) . 0.43 0.39 0.45 Dose of radioactivity (pc) 11.6 12.7 4.99 Duration of experiment (days) 4 2 2 percentage of dose C 2 in expired air 57.7 50.1 46.5 Triacetio acid laotone 12.6 13.1 23.5 Urea 0.5 0.6 1.1 Total metabolites in urine 13.1 13.7 24.6 Total radioactivity in urine 22.6 30.8 34.8 Total radioactivity in faeces 2.4 5.8 3.2 Total radioactivity in tissues 21 9 11 Total radioactivity accounted for 104 104 95 85

Table 24 Fate of II — Triacetic acid laotone in t e rat (administered , orally as an aquas solution)

Animal 12 3 Body weight 004 0,20 0.22 Dose level (mg./kg.) 364.3 266.5 273.0 (m.-mole/kg.) 2.9 2.1 2.2 Doe• of radioactivity 7.2 3..5 2.5 Duration of experitent (days) 3 3 3 percentage of demo

.002 in expired air 45.1 45.4 47.8. TriaCetio acid lactons 14.6 ::-20G1 24.3 Urea Q.2 0.3 0.3 Total metabolite in urine • 1448 20.4 24.6 Total radioactivity in urine #4.7 21.9 24.0 Total radioactivity in faeces 9.1 .6.5 4.4 Total radioactivity in tissues 19.5 8.2 10.1 Total radiciaotivity accounted for 82 86

86.

TAM 25. THE DISTRIBUTIONOf RADIOACTIVITY IN THE TISSUES OF. TWO RABBITS ADMINISTERED [140414=0p0A0ETIO ACII 0R LX

&APIA Not 1 484 medkr.) montage Tissue yet weight (ems.) Specific activity ittt Liver 54 0.0022 0.38 Kidneys 14 0.0043 0.19 Spleen 2 0.0014 0,01 Lungs 10 0.0030 0.09 Blood 235 0.0068 5.10 Intestines 450 0.0012 1.75 Skeletal muscle 610 0.001.1 2.12 Plain muscle 305 (won 1.06 Heart muscle 11.5 0.0019 0.07 0.0005 Fat 610 1 )11

b) RAtIbi i 6 (59.1mcr.Ag.)

Liver 94 0.0023 1.7 Kidneys 18,3 0.4021 0.3 Lungs 10 0.0025 0.1 Blood 203 0.0037 5.6 Skeletal muscle 580 0.0005 2.4 Heart muscle 7 0.0024 0.1 Fat 580 1 0.0002 )'2. TABLE 26. THE DISTRIFTTION OF RADIOACTIVITY IN THE TISSUES OF TWO RATS AMR SUBOUTANEQUS INJECTION W TH 1lAC4)-DEHIDROACLTIO

a) Rat No. 1 (630 mg./kEe.) Percentage suit Wet weight (gm.) pnecific.activity (wig.) 44 dose Liver 7.0 0.021 1.6 Kidneys 1.9 0.021 0.4 Spleen 1.? 0.022 0,4 Bloods Erythrocytes 10.6 0.009 - 1.1 Plasma 8.6 0.025: 2.4 Skeletal muscle 55 0.4107' 4.3 Heart Muscle 1.1 0.007 0.1. Ovary 1.3 0.007 0.1 Fat 55 0.001 , 0.6

b) RANo. 2 992.4 Pethterl. Liver 6.2 0.037 2.9 Kidneys 1.4 0.021 0.4 Spleen 1.0 0.018 0.2 Blood: Erythrocytes 6.2 0.053 4.2 Plasma 5.0 0x60 3.8 Skeletal muscle 30 0.017 ' 6.5 Heart muscle 0.5 0.030 0.2 Brain 3.0 0.011 0.4 Fat 30 0.011 4.2 2:a.1 27 THE CONVFBSION OP I !IOATIC ACID. AND HYDROXI DEMIOACETIC ACID INTO mIR Rasnonvz IMINO COIIPOUNIG IN HAMM? AND RAT MINES

Pcensge reOOVer7 from

Rabbit urine as Hat urine as Time after f : addition to i OH- imino- DNA imino-Na OH-DHL imino-...-. urine (hours OH-ItlA OH-Dna

0 95 7 101 2 101 2 , 93 2 2 96 7 97 5 102 1 95 3 4 92 5 96 i 96 6 92 6 ts 84 16 90 9 91 7 85 17 24 21 69 13 82 21 76 18 66 32 18 71, It 97 37 71 15 78 ... GliAPTER5 DiSOUSSION

The metabolic changes or dehydroacetia acid appear to be centreU exclusively around its 3-acetyl group. Biologically speaking, the most important property of the side Phoin is its ability to form Schiff's bases. Thus, dehydroacetio acid oolbines with plasma albumin and is, therefore slowly excreted, while triacetio acid lactone and imino debydroaaetic acid are both rapidly eliminated by the animal organism. This affinity of the side chain carbonyl for amino groups is further demonstrated by the formation, of imino compounds in the urines passed by a Jnos previously dosed on dehydroacetic acids by the facile combinati©n of dehydroacetio acid with amino acids (Igunhi and Bisatsune, 1957 Iguohi.ta., 1959) and by the ready formation of carbonyl derive. ire* (Perkin, 1887). Poet mortem analyses of An"171,15 dosed on 11% -dehydroacetio acid revealed that no tissue entrained more radioactivity than the blood (see Tables 251% 26). In the rat, the plasma was found to contain 1.77 times as much radioactivity as the esytbrocytes. This is in agreement with Woods. (1950) who found by a spectra- photometric method that in the dog this ratio was 1.76 and Inman 2..2.5. The depot fats were found to contain very little radioactivity; this is explained by the strong plasma binding of dahydroaoatio acid and, presumably, by its hydrov derivative. 90.

Whereas triaoetio aoid laotone is rapidly oxidized to carbon dioxide in the rabbit and rat, dehydroacetic acid and its amino derivative Show much greater biological stability. This is apparently due to the stabilizing influcnoe of the substituent in the 14 3-position. Thus 0 labelled animate, oxalate, acetate and acetone - possible degradation products -were not found in the

urines of rabbits and rata dosed on 111404.1.debydroaoetio acid or iminolehydroacetio acid, Furthermore, it is known that a

laotonase which opens the pyrone nucleus of triacetio acid laotons is without effect cadebydroaostio aoid (Meister. 1949a)" 2,6. aimwthY1.4wpyrone and its 3-carboxyl derivative are not metabolites; this precludes biological opening of the pyrone nucleus in the 1-2 position followed by rearrangement end ring closure. Vet surprisingly 140 *oroinol - formed by the action of barium hydroxide on debydroacetio aoid (Collie, 1891; Oollie and Myers. 1893, (Jarlinfanti and amain, 1911) was found to be absent in the urines of animals dosed on [:140 J -dehydroaoetio acid. 1 Since the animal organism appears to be unable to rupture the pyrone nucleus of dehydroacetio acid, therefore, it must resort to other means of attack. There is no evidence that reduction of the side chain to give a secondary aloohol °pours (3-(i-hYdronr)ethyl- triacetio acid laotons) since animols dosed on debydroacetio acid show no increase in gluouronide excretion (Shidemanst_al. 1950;

Dr. P. Gessner, private ocuminioation), Also, it was shown in 91.

the present work that the difference between free and total pyrone in the urines of animals vilmimetered delvdroaoetia acid was due to the formation of artifacts rather than to the 000urrence of pyrone conjugates. This was confirmed in a rabbit (No. 6, Table 21b)

where the difference between total and free pyrone estimated on the urine collected directly tram the bladder of the killed animal

was Shown to be within e2perimental error (ag). Complete reduction of the labile side chain does not occur since 3.ethyI-triacetio acid lactase is not a metabolite of dehydro. acetic acid. An oxidation of dehriroacetio acid was hinted at by the experi. SeeversA.A. (1950) who showed that the uptake of oxygen

ver slices increased when incubated with dehydroacetio

acid. Rat muscle and oerebrum ILIUM and kidney slices did not behave in this manner. That oxidation of dehydroacletic acid does occur was conclusively shown in the present work by the isolation of 3•glyoolloyl.triacetio avid lepton. (hydroxy.dehydroacetie acid) from the urines of rabbits dosed on the pyrone. The fact that this compound (and also imino dehydroacetio acid and dmino hydroxydehydroacetic acid) has an ultra.violet absorption spectrum almost

identical with that of dehydroaoetio acid, explains the results of Seegers ida. (1950). These workers found by a spectrophotometric method that of ingested dehydroaoetio acid, same 15.20 was excreted 92.

via the kidneys in the dog, monkey and rat. In view of the above,

this value includes hydroxy dehydroaoetio acid; furthermore, the

method does not differentiate between the free pyrones and their

imino derivatives. The speotrophotometrio results of Ileister

(1949a) must similarly be treated with some reserve; these indicate that deb,ydroacetic acid is stable in rat liver and kidney slices.

Although no experimental evidence is at present available, it

is suggested that in keeping with the attempts of the animal

organism to increase the polarity of foreign lipid soluble compounds (Brodie, Gillette and La Du, 1958; Williams, 1959), dehydroacetio acid is Ilydroxylated by the liver microsomee in the presence of TEgli and off. This would provide a more polar o=pound (hydrov dehydroaoetim acid), readily eliminated vie the kic eys. Thus, the urinary ratio of hydrov del ydroacetic acid to

ehydroacetic acid in the rabbit is about 14. and in the rat 1.5. Now, penetration by a ocespcnuad of the lipid membrane of a miorosome can only occur in the substance available either in its =dissociated form or as a complex with one or other of the normal cell constituents. Slime it is thought that the zone imraedia.tely muround4ng the microsome has a pH somewhat lower than the remaining cell contents the passage of weakly acidic substances such as dehydroacetio acid across the membrane would be possible.

The ease of formation of amino acid oomplexes with the pyrone might 93.

also explain its availability for mdcrosomal hydroxylation. Thus imino dehydroacetio acid - a compound which does not form Schiff's

bases with amino acids • is not bydroxylated in the rat. Since there is no evidence that dohydroacetic acid interferes with the active transport mechanism of the renal tubolqs (Shideman and Rem, 1951) it is most unlikely that it would enter by this mechanism. Hydrogy-dehydroaoetio acid, the first oxidation product of va dehydroaoetio acid, is no% subjected to the metabolic attacks of the animal organism. In spite of its seemingly labile 3.glyoolloyl side chain, large amounts escape destruction and are eliminated in

the urine. A. sigrAficant portion appears to be oxidized, however, since a rat dosed on hydroxy debydroacetic acid pissed urine which

on chromatographic investigation *hawed the presence of triaoetio

acid lactone. This phisnowanon is most readily explained by a gradual aide chain oxidation of 3.1313roaloyl.triaoetio acid lactose (see fig. 9). However, chromatograms prepared from portions of the urines of animals dosed on dehydroacetio acid failed to reveal the presence of the expeoted hypothetical intermediates. 9q.

Group tested for Colour reagent Reference

.0110 a) 2Z aq. p-phenylenediamine followed by 2N.acetio acid Poligl, 1956. and then 11202. (Aldehydes showed up, as blArar spots). b) malachite greendecolourised with sodium bisulphite (0.8 g. Peigl, 1956. malachite green, in 2 water containing 3 g.7 sodium sulphite).

..00.000H 0..0574 0.phenYlenediaane in lC trichloraootio acid. After • Lederer and spraying, the chromategrams were Lederer (1957) heated to 1100; (06-keto acids "'hew up as fluorescent spots).

Ig +aq. bromooresol green Feigl• 1956. (Carboxylic acids show up yellow spots ).

It iS possible that the first step.in the t o of hydrox dehydrometio acrid is catalysed by the same enzyme which converts

141actaldehyde to 42.0propaned of (Gupta aria Robinson, 1960). This system, which is TEN depenient e, is of wide occurrence and has a relatively low speoifio activity. though An Ave experiments indicate that reduotion is favoured rem Oval of the laotaldeh de would upset the equilibrium anl thus favour oxidation.: The second step in the oxidation of hydroxydehydroaoetio'actd the oxidation of an aldehyde to a carboxylic acid is avell known metabolic process (Williams, 1959). 95.

Another possible route from /lydroxy del ydroacetio acid to the hypothetical a. keto.;acid might be via an enzyme system similar to that which oatalysii the oxidation of p.nitrobenaylaIoohol. Thus, Gillette (1958) obtained oertain liver preparations which were able to nvimae p.nitrobensyl alcohol to pinitrobensoio acid. One component of this system was shown to be a DFX requiring alcohol dehydrogenase. The final two steps in the oxidation of hydroxy dehydroacetio acid are deoarboxylations. Thi41 type of reaction which *emirs with a large number of compounds in the animg1 body. It is patent Prom Table 28 that both the rabbit and the rat give rise to equal amounts of triaoetio acid laotone (formed triacetio acid laotone) by the deglyoolloylation of hydroxy delvdroacetio acid. That the urinary triaoetio acid lactone contents are different in two species is because the rat is equipped with an enzyme system which is more effective in oxidising the lactone (Witter and Stets, 1958b). In view of this, it is difficult to interpret the results obtained when[ 2 0F triacetic said laatone was fed to rabbits and rats (see Table 10). In this series of experiments' it was found that rabbits gave rise to 544% 14002 and rats 464% CO2 in the expired air. When looking for a possible explanation, it must be remembered that the rat was given about six times as much of the lactone as the rabbit: Mourkides (1959) found that when140.acatone was given in large amounts to rats, most of the ingested ketone was excreted unchanged, .'whereas at a low dosage* metabolikinoocurred to a much greater extent (also see Williams, 1959. This effect of dose Level on metabolic behaviour is also found in the case of dohydroacetio acids In the urine of a rabbit given a large dose of the pVrone (No, 4, Table 21b), about 50% of the ingested pyrone could be accounted for as dehydroaoetio acid and'itshydroxy deriVative. Howeirer* at a dose level of 1/8th of this, only about 25 of the ingested pyrone could be accounted for &a urinary dethyglioacetio and hydroxydelkydzvacetio acids. Besides a gradual oxidation of the 3.acetyl side chain of dehydroaoetic acid* a direct deacet lotion sight occur to provide triacetio acid leptons. However* there is no evidonoefor the participation of ooemayme4 since 1110..acetate was found to be absent .frogs the urines of animos dosed_on[32.041-dehydroacatio acid. Furthermore* triacetio acid laotone is not acetylated in the rabbit and rat.

Whenlsolated farosr rabbit urine, dehydroacatic acid and its hydroxy derivative are withoutoptidal activities (both compounds have unsymmetrical 5-0 atoms). It would appear* therefore, that the two compoands are attacked by enzyme systems which are optically non-specifio. 97.

Deivpiroacetic acid is an inhibitor of suocinic dehydrogenasi both in vivo soap' vitro, (Seevars,- at al., 1950). In support of Servers and his coworkers, it was found in the present work that theourites of PliohnOs dosed with [240d-dOhAreacetio- acid contained no 240-suminio acid. This indicates that the suocinuria experienced by these vitmAls is due to sucoinic acid trod natural sources rather than fray the breakdown of dehydraecetio acid. ItIiculd be of considerable interest to discover by what Mechanism dehydroacetio acid inhibits suocinio dehydrogenase. Substances which inhibit this enzyme may be classed accordlog to the following (Goodwin, 1261)s i) SH inhibitors. ii)Competitive inhibitors. iii)Compounds abelatingwith ferrous iron. iv)Compounds combining with the PAD moiety of the enzyme., The: exvide e against dohydroacetio acid acting as a general phydry ir45.bitor" is overwhelming. Thus Seeversiaja. .(1950) showed that thioglycellate,and aysteine were without effect on the inhibition. The /MO Workers demonstrated that the pyrove inereacted the ensyme activity of urease and later lechaut (1952) showed that pepsin was unaffected by the compound. Dehydroaeetio acid does net comiete with sucoinic acid since on increasing the concentration of the latter, the inhibition is not reduced. 98. .

Dehydroacetio acid inhibition is unaffected by ferrous ions (Seaver& et al., 1950). A combination of the pyrone with the free amino t• • • of the adenine moiety of the FAD residue is rather unlikely. This type of reaction would be •. expected to 'affect a large number of enzyme systems where FAD (or, indeed, anY,other adenint-oontaining co.ierimmi) is required as coenzyme. Thus ShilmmxosuldEeno (1951) .;.showed that dehydroacketio aoid was without effeot an the ATP requiring active transport mechanism of the renal tibiae*. 'Blocking of the adenine amino group reap would be expected to adversely affeat,the reabsorption of glucose and inorganiz phosphate from the renal tubules. It is suggested that dehydroadetio acid inhibits sucoinio . jek4 divse n age aei nonmocaspetitively by reacting with an aotivej 4P or .enrino group of the stiociilic dehydrogenase apoenzyme. The activation of urea.* by dehydroacetic acid night' be explained by the removal of arrovelnfa fro*& the following sYsteas 99.

,Bei the use of dehydroacetic acid_as a food2reservative. It is

evident from the present work that dehydroacetic acid is a. ,coMpouad

which is not suitable for use in human food as a preiervative or as

an additive to cosmetics or foot powders. Both the substance itself

and presumably its hydroxy derivative combine avidly with plasma

albukin and are, therefore, slowly eliminated from the'animal organism.

A further important conseenence of this would be the unavailability of both compounds for further conversion to metabolites which could then be more readily excreted.' It is.suggested that both.dehydroacetic acid and its hydroxy derivative are slowly converted to metabolite 'er and triacetic acid lactone and that both of these compounds are then readily eliminated from the animal organism.

It would, not be out of place here to consider for a few

, moments the stricture ..of metabolite "X". ultraviolet absorption data strongly suggests that the compound:' is a 3substituted 6-e ethyl.-2,

3-dihydropyran.4,4 diem (i.e. that it is of the sane species.as dehydroacetiaacidOwdroxydebydreacetic acid and their respective mine Compounds rather than triaoetic acid lactons). It is put forwards here that metabolite "X" is in fact triacetic acid lactone-3-

carboxylic acid (see fig. 9). That the. apparent pka and equivalent weight are higher'than expected for the compound might be explained 100

by assuming these values to be for a salt of the acid (where the enolic 4ydrogen is intact but the proton of the carboxyl group is substituted).

A further property of dehydroacetic acid which makes it suspect as a food preservative is its ability to inhibit succinc-dehydrogenase ice. This interference with intermediary metabolism would most certainly cause an accumulation of acidic substances and thus the severe acidosis observed in experimental animals given large doses of debydroacetic acid. iol-

FIG. 9. POSSIEZE _IMMO= PATHWAYS etio 40H11 0

C 140 are marked thus e

• • R.0 CH, I 1101210 R.00ON NH cospoundst d•bydroacetio acid) urinary artifacts.

SGCH2C11 ›- (bydrovdshydroacetio acid

direct tleacetylation? • 2.00000H

R.0001i

Rai (triaoet&o acid factore

• 3 902

3002

10 2-

TABLE 2B. Thjo relative oortanne of the sources of

In the arguients presented below, it is moused that respiratory A4002 is derived from two sources 91)1r,, namely by the decarboxylation of tim hxpothetiaal intermediate P4C3,1-TAL.3-4000/1 and the oxidation of (.4403.1..TAL (see also fig. 9):-

[145].TAL.1400OR formed [140 J-TAL 314002 (a) (a-0/3) 14002 (°/3)

urinary [140 1.TAL (b)

Nom, If the total respiratory 14002 is expreseed by a the urinary (14031.TAL and the formed [140.0..TAL (where a, b and a represent percentages of the radioactivity fed). zaa the reapiratory CO2 derived from the deaarboxylation of [ 1..TAL.- 1400011 is ci.3 ADA the respiratory "002 derived from the degradation of lad of the formed [ j--TAL is (a-.0/3)

It is clear that a b + (a - 0/3) or a 3/4 (a + b) (1) using formula (1) it is pose ble to calculates i) the formed (1403)..TAL (a) ii)the respiratory-14002 derived from the deoarbovlation of [14033 TAL,..14000H (0/3) muff*. the oxidation of 403 ji.TAI. (a - (1/3).

/03

AlaAafg Rabbit 1 7.2 1845 19.3 6.4 0.8 2 7.9 12.2 3.51 5,;0. 3 10.4 . 8.8 . , . . 1 4 54 1.3.. . . 4 2 34 ' 4.0 0.7 5 9.8 7.1 12.7. . 2 ' 5.6

6 li.h .1 1,0 15.3 5,,A, 10.3 Rat 1 25.8 1.3 20.3 6.8 19.0 2 7.9 13.9 16.5 5.5 2.4 . , 3 14.3 2.1 1305 • ' 4.5 - 9.8 4 12.6 143 10.5 3.5.. 9.1 5 10.9 1.0 8.9 , 3.0 7.9 6 13.6 1.4 11,4 3.8 9.8 1* 10.4 0.7 8.3 2.7 7.7

* In this all administered t5r subcutaneous injection. nr4i 4 PHYSICAL PROPERTIES OF CERTAIN PIRO=

1. TAtravlolet Absorption Spectra These were measured with a phlegm spectrophoto-, meter SP 500 in ethanol 0.1N4H01 (------) and 0.111-N40H

Apparent t K Values aid Eguticalq4 WeAght Solutions of the pyrones were made up in freshly boiled distilled water and titrated against 0.02N-NaCH using a Cambridge Bench Type pH Meter.

Qqtical Hot4ion .Aquas solutions of MA, OH-DHA, NH6DHA, :Nlig)H-DHA, and metabolite X showed no optical activities. 29 LIGHT ABSORPTION OP S0161 MIMS

COMPOUND 0 RC' 0. - NOR ETRATCOL , 1 t t • Amin I mi Amaxim., xx Amin fad Amaxf maxl )qstin ''adaz 1 m 0 M z10 M X 10-.'" M M 10 ' m x 10--1 93 X lej

• ' 1 I 7 ; Debydroaoatio sofa 223 12.9 - 259 1.3 230 14.9 261 3.4 222 17.5 260 3.4 305 12.6 292 8.6 303 13.2

Hydroxy- 222 9.6 253 1.3 231 21.5 262 4.7 224 11.3 260 0.8 dehydroacetio said 309 9.2 296 11.0 308 10.2

Triaoatio acid 8.3 242 2.1 277 B-5 248, 47- 210 8.7 238 1.6 leetone ''', 5.5 284 6.9 . . Imino-delvdro- 232 416r11- 253 2.0 233- 19.4 261 5.0 iiet- 4,.:8- 253 2.0 acetic acid 302 12.1 295 .471 301- *34 /4•0 8'7 302 PO Imino-bydroxy- 232 16.6 253 2.5 233 19.4 26t 4.9 229 15.0 253 2.5 dehydroa.oetie acid 302 12.8 295 9.1 301 13.9 i , Metabolite X 220 9.3 255 1.5 236 13.2 267 3.6 223 8.5 267 2.1 (mo leoular weight 319 9.6 310 5.9 321 • 237) 9.5

ft If /"C", TP fS at./

26 of!, rg CET? c Rei f) 1 Fr CTois, Co a ZW/ivo - 3EWYPile*IfincEric 17c0

3 K7,1> r fwvo )•"-- iCH )lq" (.4 'I it I o 9

TABLE 30 APPARENT DIC VALUES ATM

NEUTRAT,,BCFIVALENTS OF CERTAIN PirljOIES

gammai ga Eauivalent Reference

Dehydroacetic acid 3.28 .. Oswald, 1889 5.3 172 Berson, 1952 5.3 176 present work - Triacetio acid lantana 5.0 129 Berson, 1952 4.95 131 present work Ifydrowdebydroacetio 1 5.0 180 present work acid

Metabolite "X* 4.9 237 present work I/0

4PPEzigi A

THE RATES OF EXCRETION OF CO2 IN THE EXPIRED AIR AND TOTAL URINARY ACTIVITIES FROM RABBITS AM RATS DOSED,ON

[14C4j-DEBIDROAOETIC ACID, [14c3).TRIACETIC ACID LACTONE OR [1404)-IMINO DEHYDROAOETIC ACID. rn

TABLE 31 TOE 'EXCRETION OF 24C0o IN THE EXPIRED `AIR OF RABBITS ADMINISTERED 114;41-DERYDROACETIC ACID OR.ALLX

(a) jabbit Na. 1 (18.4ned6e) Tim' (.hours) 4 dose total % dose A 400041r. 4 0469 0.68 0.17 8 1.26 1.94 0.31, 12 1.32 3.26' 0.33 16 - 1.4 4.67 0.35 20 0035 5.52 0.21 24 0.62 6.14 0.15 32 , 0.52. 6.66 0.1.3 45` 0.57 7.23 .0.14

(b) Rabbit Nat 2 (78.5 1Ata./Ite4 9 2.24 2.24 0.25 21 2.82 5.06 •0.24, 27 .1..01 .6.07 0.16 33 0.28 6.35 0.03 45 0.47 0.82 0.04 . 51 '9447 7.29 0.08 57 : 0.58 . 7.87 0.10

(0) PO (60.grlizpike.1 6 1.45 1.45 0.24 15 2.00 3.45 0.22 23 1.78 5.23 0.22 40 2.25 7.48 0.13 48 0.32 8.00 0.07 65 0.35 9.35 0.02 86 0.50 9.85 0.02 112--

TABLE 32. THE EXCRETION or ihr.,92 IN TEE E IRED AIR OF RATS ADMINISTLRED 1 DEHYDROACETI0 ACID ORALLY

a) pat No. 1 (63.0 ma./}ca.) Tion, (Wpm') iii8Mit toptal %Acme $ dosa4r. 6 3.86 3,86 0.64 12 9.70 13.56 1.61 24 5.81 19.37 0.48 30 1.66 21.03 0.29 36 0.90 21.93 .0.15 48 1.42, 23.35 0.12 54 0.36 23.71 0.06 60 0.81 24.52 0.13 72 1.12 25.64 0.93 96 0.16 26.80 0.01

b) Rat 1 kb . A (17.6 cedlp.)e 18 4.4 4.4 0.24 24 1.6 6.0 0.27 42 1.9 _ 7.9 0.11

;tat No. 3 (132.0 ag./ka.) • 24 8.4 8.4 0.35 48 1.6 10.2 0.07 72 4.1 14.3 0.17 d) 40 5 1.58.11 ifuthAse.) 24 9.6 9.6 0.40 48 0.9 10.5 0.02 72 0.3 10.8 0.01. 96 0.1 10.9 0.01 "3

TABLE 33. THE EXCRETION of C0 N THE EXPIR,BRID AIR OF Rug ADMINL.§VERED [ MUD ACETIC ACID BY SUBCUTANEOUS MOTION ftat No. 3. (63.8 meilka.) il,ma (hrs.) 4fl8, tom. % dope % doseAr, 3 1.1 1.1 0.37 6 1.8 2.9 0.60 22 4,5 7.4 0.2e 48 2.8 10.2 0.11 72 0.2 10.4 0.03.

TABLE 34. THE EXCRETION OF WTAL RADIOACTIVITY N THE NE 0 NIS D 4 DERYDROAC4TV ACID "ITBCDTANEOUS INJECTION

Rat Not ,j. (634 isodikg,) Tilliq (I1113-) Vp?.I.ve (m1.) %,_dose totel % dose 23 4.7 13.5 33.5 48 6.2 11.8 25.3 54 3.0 2.6 27.9 72 35.0 5.3. 33.0 TABLE 35. THE EXCRETION OF TOTAL RADIOACTIVITY IN THE URINES OF RABBITS ADMINISTERED Li4..61.EHYDROACETIC ACID ORALLY a) 13abbit No. 1 (18.4 mg.ika.) Tame thrs.) Volume (ml,) % AV" total % dose 11.25 200 35.6 .35.6 20.25 200 15.2 50.8 32.25 100 5.6 56.4 46.5 157 7.5 63.9 52.5 114 0.8 64.7 58.0 114 3.2 67.9 93.0 137 5.3 73.2 b) *Mat No. 3 (10.7 mq.Acg.) 48.0 27.5 3.39 3.39 48.5 24.0 4.45 7.84 51.0 100.0 21.20 29.04 79.0 15.0 0.64 29.68 119.5 33.0 3.49 33.17 120.5 46.0 4.55 37.72 121.5 104.0 9.86 47.58 132.5 75.0 2.54 50.12 137.0 207.0 1.91 51.03

Table contd. „ Continued from overleaf

TABLE 35. THE Ey9RETION OF TOTAL RADIOACTIVITY IN THE URINES OF RABBITS .914INISTERED tLD...WDROACETIC ACID ORALLY c) Rabbit No. 4 (491. mr.Ae.) Time thrs,1 Volume (m1e) ILACILI total. % dope 48 250 41.3 41.3 96 240 23.5 64.8 120 113 1.7 66.5 168 320 2.4 68.9 240 175 1.1 70,0

) 1400Pit 2 . (60 mizake.) 15 11O 14.1 14,2. 23 60 10.8 24.9 40 250 25.0 49.9 64 140 7.1 57.0 88 110 5.2 11,3 55 2.2 :::: 162. 340 6.9 71.3

116

TABLE 36. THE EXCRETION OF TOTAL RADIOACTIVITY THE URINES OF RATS ADMINISTERED l'ACA)-DERYDROACETIC ACID ORALLK

a) REV4 k2. 1- (63.0 114.1'a.) Time (bra.) Volume ,(m10) Lam total i dertt 24 14+5 22.46 22.46 48 13.2 15.04 3(.50 72 10,5 4.45 41.96 96 200.0 3.24 45.22

b) Rat Jo. .2 (1716 lust ¢,1 18 12.0 15.7 15.7 24 16.5 9.2 24.9 42 12.0 14.3 39.2 66 23.5 7.7 46.9 96 25.5 3.8 50.7

o) Apt No. 1 (112.0 mff.4m.) 24 5.8 16.2 16.2 0 4.8 12.5 28.7 72 5.2 6.8 35.5 98 11.0 4.2 39.7

Rat 6 (6q,0 mpilig.) le 7 10.8 10,8 24 4 MO 20.8 48 7 7.5 28.3 96 3 1.0 29.3 JP1

TABLE 37. 1 3'40 IN WIRED AIR OF A RABBI ADMI TE,RED [M,a TRIA TIC A ID TONE 0

Rabbit qp. 1 (53.6 zazikg.

Time_ (bra.) LAM total % doss, dose/hr. 4.0 2.3.4 23.4 5.85 20.5 29.1 52.5 1.76 29,5 1.8 54.3 0.20 47,0 .3.4 57.7 0.19

TABLE 38 y#E ) XgRETION OF TOTALOACTIVITI URINE 9r A RABIAT Itama TERED ETIC AC D C

Aabbit No. 3. (55.6 B9z./kg.)

Um* (hrs.) Volualt 04.) $_dosit total % close 3,0 4 4.1 4.1 20.5 25 17.1 21.5 26.5 3.5 0.8 22.3 69.5 13 0.3 22.6

lit

TABLE 39. E:t - OF AIR TIK RATS TERED TRIAOETIC ACID 'AMORE oRAlai

a) Rat No. m

Time (pre.) /Ant total % dgatt $ dosehrt 24 44.5 44.5 1.85 48 0.9 45.4 0.04

b) Rat No. 3 12'73.0 6 28.4 20.4 4.77 18 19.4 47.8 1.08

TAB 40 . THE EXCRETION OF TOTAL RADIOACTIVITY URINE OF A RAT ADNINLSTEByED TRIVETV ACID 1.4CT0IS ORALLY

Rat. No? 1 (34413 Era/kg.).

Time (hra.) Zaan (m1.) $ dose total % dose 30 2.9 9.7 9.7 48 4.2 443 14.0 72 2.5 0.7 14.7

//9

TABLE 41. TO EIORETION OF TOTAL luoioAcTivrrr N THE UR111 OF A RAT ADATNISTEHED fA, 41.-D4II/O-DEHXDROAGETIC ACID pliallX

Peit Not 2. (2374 mft.4cg.)

Time 04.06) Ygileums 414 Liket total % dome 24 3.9 8.0 8.01 30 5.0 12.36 20.37 48 3.2 12.36 32.73 52 170.0 28.21 60.94

TAKE 4. XOFZTION OF 3400 N THE

Pat No. 1 j229.6 ti2.Akg,) reins OMR) citt does X dowahr.

5.0 0.5 0.5 0.10 22.5 1.5 2.0 0.08 46.5 0.1 2.1 0.01 /,2o

APPENDIX III

DIETS

Diet No. Lib (Dixon Ltd., Ware, Herts.).

Wheatmeal 47% Sussex ground oats 40% Fishmeal 8%. Dried skimmed milk 3% Dried yeast" 1% Sodium chloride 1%

WAt No. j1,8 (Associated London Flour Millers).

Dried grass meal 30% Barley meal 20% Bran 15% Ground nut 15% Linseed cake 10% Dried meat and bone meal 8% Sodium chloride 1% Calcium carbonate 1%

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