Demethylation in the mammalian metabolism of N, N-dimethylurea

Gen Pharmac ,

1975, Vol 6, pp 275 to 280

Pergamon Press. Printed m Great Britain

DEMETHYLATION IN THE MAMMALIAN METABOLISM OF N, N- DIMETHYLUREA MOHAMMADS. DAR* Department of Pharmacologyt, FaUlty of Science, Mahldol University, Bangkok, Thailand Abstract--1. The metabohsm of N,N-dlmethylurea has been investigated m the rabbit and the rat, and ewdence has been presented to show that the compound undergoes stepwtse demethylatmn whtch leads to the formatmn and excretmn of urea and methylurca. 2 According to McKenzle and du V~gneaud the source of urinary urea carbon is the metabohc carbon dmxlde: they found that the ratm of specific actiwty, u r e a / C O s , approached one, during the same interval of time, after the oral administration of L-[methyl-t4C]methmnme m rats. 3 The slgmficant increase m the ratm of specific actw~ty, urea/respiratory CO~, m animals rejected mtraperRoneally wRh N,N-dlmethyl[x4C-carbonyl]urea indicates a metabohsm of dimethylurea to urea wa methylurea which ~s accomphsbed m such a manner as to maintain the integrity of one or more of the mtrogen carbon bonds of dlmetbylurea 4 However, the data obtained do not permR the exclusion of either ammonolysis or dtrect demethylatmn as a parUc~patmg factor m the demethylaUon of dtmethylurea. INTRODUCTION

Rochester, New York. Jack bean urease powder (EC 3 5 1 5) was purchased from Worthington Blocbemlcal THE POSSIBLE occurrence of methylurea and other Corporation, Freehold, New Jersey The ion exchange alkylureas in the urine of mammals was the topic resins, A-641(OH-) and C-244(H+) manufactured by of considerable investigation and discussion in the Ionac Chemical Co, and purchased from J. T. Baker years 1875-1885 (Salkowski, 1877; Schiffer, 1881; Chemical Co, Plulhpsburg, New Jersey, were of 16-50 Schmideberg, 1878). Otto Folin (1907) was the first and 30-80 mesh size respectively Dimethylaminehydroto suggest their possible occurrence in the human chloride was also purchased from J T Baker Chemical urine, especially after the ingestion of a highly Co, Phlllipsburg, New Jersey. Selas porous porcelain crucibles (Cat. No. C-9185) were obtained from the nitrogenous diet. During the course of our investigation of the Scientific Glass Apparatus Co., Bloomfield, Now Jersey. The rabbR metabohsm chamber was purchased from the metabolism of methylamine and methylurea (Dar Plastic Manufacturing and Supply Co, Lansing, Michie t al., 1969; Dar, 1970), it was thought interest;ng gan Potasslum[a4C]-cyanate (sp. act. 4 31 mC1/mM) to study the metabohsm of dtmethylurea as it might and [t4C]methylanune hydroehlorlde (sp. act 5-74 also be involved in the metabolism of methylamine mCl/mM) were purchased from the New England since the latter is known to give rise to dimethyl- Nuclear Corp, Boston, Massachusetts. L-[methyl-t4C] amine (Asatoor & Simenhoff, 1965) which, mcthionme (sp. act 55 2 mC1/mM) was obtained from like methylamine, may change metabolically into the ICN Corp, Irvme, Cahforma. Methyl[t4C-carbonyl] dimethylurea. In addition, following the demon- urea (sp. act. 0.49 mCI/mM) was synthesized (Dar, 1970) stratlon of a metabolic interrelationship between in the Department of Pharmacology, Medical College of Virginia, Virginia Commonwealth University, Richmond, methylamine and methylurea (Dar, 1970), it was Virgima, U S.A. deemed important to look for a similar interPaper chromatography was carried out on Whatman relationship between dtrnethylamine and dimethyl- No. 1 paper at room temperature. Paper chromatograms urea in an attempt to suggest a general metabohc were developed m solvent system A (chloroform-ethanol: interrelationship between amines and corresponding 98: 2% v/v) and B (Perry and Schroeder, 1963) ureas The present investigation deals with the (acetomtrile-fornuc acid-water: 80: 2: 18% v/v/v) by study of the metabolism of N,N-dimethyl the descending method. As a locahzmg agent p-dimethylarmnocinnamaldehyde (5 % m diluted sulfuric [t'C-carbonyl] urea in the rabbit and the rat. acid) was employed in some cases. Developed radlochromatograms were scanned by passing the paper MATERIALS AND METHODS strips through a Packard Radlochromatogram Scanner model 7201. Rf values for methylurea and N,N-dlmethylDlmethylurca was purchased from K & K Laboratories, Inc, Plainwew, New York Methylurea and potassmm urea using the solvent system A were 0 05 and 0.48 cyanate were obtained from Eastman Orgamc Chemtcals, respectively. The urea almost stayed at the origin m thin system. In the case of solvent system B, Rf values for urea, methylurea and N,N-dtmethylurea were found to * Present address: Department of Pharmacology, Medi- be 0 52, 0 66 and 0-74 respecUvely. The melting points cal School, Pahlaw UmversRy, Shiraz, Iran. of isolated methylurea and methylurea p-toluenesulfot Part of work carried out m the Department of Pharma- nate were deterrmned on the hot stage using a F~sbercology, Medical School, Pahlavi UmversRy, Shiraz, Iran. Johns melting point apparatus. 275

276

MOHAMMADS. DAR

by lntraperltoneal administration and were placed in Radioactivity was measured in a Packard Liquid plextglass metabolism chambers for the collection of Scintillation Spectrometer model 578 The counting 24-hr urine A 10 ml aliquot of 24-hr diluted urine efficiency of the scintillation counter was routinely deter(pH 6-63, clarified by centrlfugation, was treated with mined and ranged between 7 8 - 8 4 ~ , and the radio100-102 mg methylurea, previously vacuum dried overactiwty of the samples exhibited little or no variation at mght (15 hr) at 55°C The urine was processedthroughwet a gwen time Isolated compounds were counted in scintillation solution I (2-(4-1-butylphenyl)-5-(4-blphenyl) A-641 (OH-)(200ml)anlonlcexchange resin The washing of the column was continued for 3-4 hr until the effluent -1,3-oxldiazole), (7g) in one liter of solution of toluenecontained no radio activity About 1 5 1 of the effluent were ethylene glycol monomethylether (2" 1 by volume) then poured through wet cationic ion exchange resin Animal respiratory carbon dioxide was collected in solution 1I (Jeffay & Alvarez, 1961), monoethanolamme- C-244 (H +) (160 ml), and the column washed with disethylene glycol monomethylether 1 . 2 by volume) tdled water Only those fractions were collected which Ahquots of 4 ml were added to 15 ml of solution I for contained radioactivity significantly higher than the background (total volume 5 1 ) The combined fractions counting Synthesis of N,N-dimethylurea Using a modification thus obtained were evaporated under vacuum, to yield dry residue containing mainly urea, methylurea and of the procedure of Wurtz (1851), dimethylamme hydrounchanged dlmethylurea The residue was extracted with chloride (408 mg) was treated with potassium cyanate three 15 ml portions of warm tetrahydrofuran The com(405 mg) m an aqueous medium (8 ml) The aqueous bined tetrahydrofuran extracts were evaporated to drysolution was then evaporated to dryness in a water bath ness in a stream of nitrogen and the residue, consisting The dry mass was extracted with hot absolute alcohol and mainly of methylurea, was then incubated with urease the alcoholic extract was also evaporated to dryness enzyme in order to hydrolyze the traces of urea The An alcoholic solution (5 ml) of dry mass was spotted on residue with 1 ml of phosphate buffer mixed with 2 ml Whatman No 1 paper (4 × 24 in size) and developed in of urease powder (approximately 10rag, activity 2 solvent C (butanol-water) by the descending method for Sumner units) was incubated for 4 hrat 37°C in a Dubnoff48 hr The solvent end of the chromatogram was sermetabolic shaking incubator rated so as to facilitate the collection of the butanol At the end of incubation, the mixture was centrifuged fraction containing the product Dlmethylurea thus and the supernatant evaporated to dryness in a stream of obtained was recrystalhzed from absolute alcohol and nitrogen The residue was extracted with three 8 ml showed a m p of 18ff-182°C portions of warm tetrahydrofuran and the combined Synthesis of N,N-dimethyl[14C-carbonyl]urea Dlextracts were evaporated to dryness The crude methylmethylamine hydrochloride (81 5 mg) was treated with urea thus obtained was dissolved in 1 ml of absolute potasslum[~4C]cyanate (81 nag) in 5 ml of distilled water alcohol Subsequent recrystalhzations were carried out and the reaction mixture was subjected to the same treatfrom absolute alcohol to obtain methylurea of constant ment as described above to yield the desired product The specific activity, m p 101-102°C (hot stage) product (m p 180-183°C) was re-crystallized repeatedly Preparation of methylurea p-toluenesulfonate Twentyfrom absolute alcohol and vacuum dried at 60°C until it six mg ofp-toluenesulfomc acid to a solution of 10 mg of arrived at a constant specific activity The purity and the methylurea in 2 ml of hot absolute alcohol were added homogeneity of the synthesized compound was checked The reaction mixture was concentrated in a water bath by paper chromatography, and passing the paper strip with continuous stirring and evaporated to dryness in a of the developed chromatogram through the radiochromastream of nitrogen The dry mass was dissolved in a togram scanner Only one radioactive spot corresponding minimum volume of 2-propanol and cooled in anicebath m R f value (0 74" Solvent ]3) to the authentzc N,NBy the addition of n-hexane and scratching, the product dimethylurea was observed was precipitated Centrifugatlon, followed by washing Female New Zealand rabbits weighing 1 5-2 6 kg were of the product with ether, yielded a cleaner product used throughout this investigation and were obtained Further recrystalhzatlons from 2-propanol-n-hexane, from the animal center of the Faculty of Science, Mahidol followed finally by vacuum drying at 55°C, were carried University Female Wistar strain rats from the same out to obtain a product of constant m p 135-136°C source weighing 150-200g were also employed The (capillary) ammals were maintained on a diet of Purina chow and Analysis++ CsH14SN~O4 (246 33) water Compounds were administered in aqueous Calculated C43-90~o H 5 6 9 ~ Nl138~ medium (3-5 ml to rabbits, 0.5-1 ml to rats) by intrapenFound" C4397~ H571~ Nl126~ toneal rejection The experiments were continued for 24 hr. During this time the rabbits were housed in a Preparation of p-toluenesulfonate of methylurea plexiglass metabolism chamber with a fecal-urinary isolated from rabbit urine Methylurea (3 478-4.388 rag, separator Glass metabohsm cages were employed for specific actwJty 8 9 × 10e and 9 2 × 10a dpm/mM) was the rats in which a stainless steel wire screen was used to treated in hot absolute alcohol with p-toluenesulfomc separate the urine from the feces, but some cross conacid (approximately 10-13 mg) and the product was tammatlon was unavoidable. During the experiment the obtained as described under preparation of methylurea ammals were kept without food and water Respiratory carbon dioxide was entrained m the stream of alr which p-toluenesulfonate The constant specific actwities of the salt in each case were 7 8 ~ 10~ dpm/mM respectively passed through a concentrated sulfuric acid trap and Isolation of methylurea from rat urine Two female finally into towers of solution II at a rate ranging between WIstar rats weighing 188-197g received 15 81 mg/kg 1700 and 1800 ml/mm Isolation ofmethylurea from rabbit urine Two female rabbits weighing 2-47-2 60 kg received 6.1 mg/kg (acti++ Analysis camed out by Spang Microanalytlcal Labvity 91 1-95 6 ~tCl) of N,N-d~methyl[~C-carbonyl]urea oratory, Ann Arbor, Michigan, U S A

Mammalian metabolism of dimethylurea (activity 17.9-18 7 gCi) of N,N-dimethyl[UC-carbonyl] urea by mtrapentoneal lnjectmn and were placed in glass metabolism cages for the collection of 24-hr urme. A 100 ml aliquot of 24-hr diluted mane (pH 6 6), clarified by centnfugatlon, was treated with 100-103 mg methylurea, previously vacuum dried overnight (15 hr) at 55°C. The urine was processed through 100 ml wet anlomc exchange resin followed by 80 ml of cationic exchange resin The fractions collected from the latter column were those possessing radioactivity significantly higher than the background (total volume about 41.) and processed in the same manner as described under isolation of methylurea from rabbit unne. Preparation of p-toluenesulfonate of methylurea isolated from rat urine. Methylurea (3.875-4.439 rag; specific actwRy 1-994 × l0 s and 1 840 × l0 s dpm/mM) was treated m hot absolute alcohol with approximately 10-15 mg ofp-toluenesulfonle acid and the product was obtained according to the procedure described under preparation of methylurea p-toluenesulfonate. The constant specific activities of the salt were 1.784 × l0 s and 1 635 × liP dpm/mM respectively. Respiratory carbon dioxide and urea formation from

277

the metabolism of N,N-dimethyl[x~C-carbonyl]urea. Three female rabbits weighing 2.45--2 60 kg received 6.10 mg/kg of N,N-dlmethyl[t4C-carbonyl]urea by lntraperitoneal administration. A 1 ml aliquot of the monoethanotamme solution which contained all of the radioactivity of the expired air during 24 hr was taken from the scrubbing tower in each of the three experiments and treated with 2 ml of saturated aqueous solution of barium chloride. The lightly stoppered nuxture was allowed to stand in the boiling water bath for 10 min, treated with 20 ml of hot distilled water and the supernatant removed The precipitated barium carbonate was washed with two portions of hot distilled water (20 ml each) and transferred to a Selas porous porcelain crucible with the md of hot absolute alcohol and dried to a constant weight at ll0°C. A 1.0-1.1 ml aliquot of the 24-hr diluted urine was placed in a 50 ml round-bottomed flask and for removal of dissolved carbon dioxide adjusted to pH 1-2 by the addition of 6 N hydrochloric acid The aliquot was then adjusted to pH 6 6 by the a d d m o n of 5 mi of potassium phosphate buffer (0.5 M; pH 6.6). Nitrogen was flushed into the solution through a glass wool filter and into two

Table 1. Excretion of radioactivity by female New Zealand rabbits and female Wistar rats after the intraperitoneal administration of N,N-dlmethyl[X4C-carbonyl]urea Per cent administered dose of t4C Species Rabbit a Rabbit" Rat a Rat c

Dose 2251xg/kg 100mg/kg 225~tg/kg 100mg/kg

Activity (~tC0 240-3.49 1.76-1.21 0.21-0.25 0-20-0.24

(2 hr)

Respiratory COs (4 hr) (6 hr)

Total (24 hr) Urine

CO2

0.525:0.17 2.195:0.23 3.854-0.23 8.154-0.22 d 0-065:001 0 185:0.04 0.585:0.05 4.16+0.21 ~ 5.094-0.40 7.955:042 9.554-0.48 1163=t=0.54f 0.614-0-05 1-585:0.59 2.914-0.79 5.654-0.11 s

Sum

72.734-1.81 80-885:1-79 80 56+2.64 84-725:2.63 84.335:1.50 95-965:1 34 89-634-2.54 95 364-2.63

Data are expressed as per cent ( + s t a n d a r d error of the mean) of admlmstered dose of t4C. a. Five animals, d. Sigmficantly different from e and g (P<0.001) b. Eight animals, e. Significantly different from d and f (P<0.001). c. Seven animals, f. Significantly different from g, d and e (P<0.001). NB. Similar to methylarmne and dlmethylamme, very httle radioactivity was suspected in the feces and so it was considered not essential to deterrmne fecal radioactivity.

Table 2. Specific actiwty of carbon dioxide and urea produced by female New Zealand rabbits after the intrapentoneal administration of x~C-labelled compounds Specific activity dpm/mM Animal weight (kg) 2"26 2"45 1.87 2.00 1"83 2.35 1.90 1.70 1"85 2.48 2.60 2-45

Compound adrmnistered

Methyl [14C-carbonyl]urea (972 ltg/kg) [t4C]methylamine hydrochloride (212 Ilg/kg) L-[methyl-UC]methlonine (370 I~g/kg) N,N-dlmethyl [t4C-carbonyl]urea (6"10 mg/kg)

Ratio Sp. ac. urea

Respiratory COt

Urinary urea

Sp. ac. COa

1"19 × lip 1"16 × lOs 1"23 × l0 s 3-52 × liP 3.52 × 104 4"30×104 5"49 × lip 5.37×104 5.29 × 104 1"40 × liP 1"79 × 104 1.39 × 104

2.21 × 105 2"27 × 105 2-49 × lOs 3"45 × 104 3.62 × liP 4"15 × lip 5.37 × lip 5.13×104 5"34 × 104 5.85 × l0 s 6.79 × lip 5 94 × 106

186 195 202 0.98 1.03 0-96 0-97 0.95 10l 40"4 37-8 42.7

278

MOHAMMADS DAR

tubes, each containing 5 ml of ethanolamine--ethylene glycol monomethylether ( 1 . 1 v/v), and into a final Ascante trap After the addmon of urease (15 nag in 5 ml of potassium phosphate buffer; activity 6 Sumner units) by means of a syringe and needle through the stopper of the reaction vessel, flushing wah nitrogen was continued for 4 hr, a time found sufficient for complete (90-94 ~o) hydrolysis of the urea Hydrochloric acid was then introduced through the needle and the reaction mixture, now at pH 1-2, was further flushed with mtrogen for 30 mln The ethanolamlne traps, after the removal of an aliquot for counting, were treated with 2 ml of saturated barium chloride solution as described above The urea radloacttvlty amounted to approximately 23 ~ of the urinary rad~oactlwty m the case of three ammals The ratms of the specific activity, urea/respiratory C02, are shown in Table 2. Respiratory carbon dxox~de and urea formation from the metabolism of other x4C-labelledcompounds Methyl [14C-carbonyl]urea 972pg/kg, L-[methyl-~4C]methiomne 370ttg/kg, and [~4C]methylamme hydrochlonde 212 lag/kg, were each admunstered lntrapentoneally to a group of three female rabbits weighing between 1-702 60 kg The experiments were carried out m exactly the same manner as described under respiratory carbon dioxide and urea formation from the metabolism of N,N-d]methyl[~C-carbonyl]urea.

B

A

0r,g;, Fig I Scannmg of radlochromatogram of concentrated neutral fraction of urine of rabbit injected with N,Ndimethyl[~C-earbonyl]urea A Urea; B Methylurea; C Dlmethylurea (System; Chloroform Ethanol, 98 • 2 ~ v/v). B

RESULTS AND DISCUSSION Qualitatively, the data obtained are similar in both species. The excretion of radioactlwty in the expired air appeared to be rapid during the first few hours of the rabbit experiments. Almost 50~o of the radioactivity excreted as 1~CO2 was detected during the first 6 hr and the remainder excreted rather slowly during the remaining 18 hr as seen in Table 1 In comparable experiments using rats, over 80 ~o of excreted radioactivity was detected during the first 6 hr; the ~4C-activity appearing in th~s case at a rate which is significantly higher than in the case of the rabbits. At each time interval (2, 4, 6, and 24 hr) the rate of 14C-excretion in the expired air was found to be sigmficantly greater in the case of the rats than in the rabbit as is also evident in Table 1. At 100 mg/kg dose level, the excretion of radioactivity in the expired air was significantly depressed as compared with animals injected with 225 ~tg/kg dose This significant depression was noticed at all time intervals during the 24-hr experiment and could also be seen from the data in Table 1. However, in all the experiments in both species, the excretion of ~4C-activity in the 24-hr urine exhibited little variation. A n average of 75 and 86 ~o of the radioactivity appeared in the 24-hr urine in the rabbits and rats respectively. The scanning data of the developed paper chromatograms are shown in Figs. 1 and 2 Peaks were identified by comparison with the Rf values of the authentic samples of urea, methylurea and dmaethylurea which were spotted on the same strip along w~th the concentrated neutral fraction of the 24-hr

Or,gin

Fig. 2 Scanning of radlochromatogram of concentrated neutral fraction of urine of rat injected with N,Ndlmethyl[14C-carbonyl]urea. A Urea; B Methylurea, C Dnnethylurea. (System; Chloroform. Ethanol, 98 . 2 % v/v) animal urme. Table 2 shows the data concerning the speofic activities of urinary urea, respiratory CO2 as well as the ratio of the two, m the case of rabbits injected with methyl[14C-carbonyl]urea (972 lag/kg; sp act. 0.49 mC1/mM, [14C]methylamine hydrochlonde (212 lag/kg; sp. act. 5 72 mCi/mM), N,N.dimethyl[14C-carbonyl]urea (6 10 mg/kg; sp. act 0.53 mCI/mND, L-[methyl-~C]methionine (370 ~tg/kg; sp. act. 55.2 m O / m M ) The ratio of speofic activtty, urinary urea/respiratory CO~, was almost one in the case of [~4C]methylamine hydrochlonde and L-[methyl-x4C]methionine treated anunals whde it approached nearly 200 and 40 in the case of animals injected wlth methyl[l~C-carbonyl]urea and N,N-dimethyl[~'C-carbonyl]urea respectively. Table 3 shows the amount of methylurea excreted in the 24-hr urine of rabbits and rats after the rejection of N,N.dimethyl[1,C-carbonyl]urea respectively. It is evident from this Table that the methylurea fraction m the 24-hr urine constitutes the major urinary metabohte of dtmethylurea comprising about 67 % of urinary radioactivity m both species

Mammalian metabolism of dimethylurea

279

Table 3. Recovery of N-methyl[X~C]urea by isotopic dilution from 24-hr urine of female rabbits and rats injected intraperitoneally with N,N-dimethyl [l'C-¢arbonly]urea', ~

Species Rabbit Rabbit Rat Rat

Animal weight

(rag)

2.478 kg 2.600 kg 197 g 188 g

15.13 15.89 3.115 2.972

(pCi)

Administered radioactivity recovered as Methylurea ( ~

Urinary radioactivity as Methylurea (%)

91.16 95.65 18.77 17.90

48.85 52.15 45.27 48.22

67.11 70.15 67.35 63-17

Dose

a A dose of 6.107 mg/kg (activity 91.16--95.65 gCi) was admintstered to each rabbR. b A dose of 15.81 mg/kg (actwity 17.90-18.77 laC0 was admimstered to each rat. The depression in the amount of z'CO2 excreted per unit time in the animals mjected with 100 mg/kg dose of N,N-dimethyl[x~C-carbonyl]urea (Table 1) could be due either to a feedback inhibition or repression of enzyme syntheses. The biochemical and genetic analysis of numerous biosynthetic pathways has led to a general concept that the concentration of the ultimate end product governs the rate of end product formation (Stadtman, 1970). In the case of feedback inhibition, the end product usually inhibits the enzyme that catalyzes the first step in the metabolic and/or the biosynthetic pathway or stabilizes the enzyme's conformational state at a lower catalytic potential, i.e. enzyme with a lower turnover capacity. In the case of feedback repression, accumulation of the end products leads to inhibition of the synthesis of one or more of the enzymes involved in its metabolic or biosynthetic pathway. The results shown m Table 1 indicate that dlmethylurea cannot be considered as being metabolically inert. The appearance of x~C-activity in the expired air first suggested a hydrolysis of dimethylurea, perhaps under the influence of bacterial nreases such as some Bacilli and Mycobacteria However, the urea fraction in the urine of rats and rabbits treated with carbonyl-labelled dlmethylurea was found to be considerably enriched with a4C-activity, which appears to have arisen directly from dimethylurea. This observation suggests a stepwise or progressive demethylation instead of hydrolysis of dimethylurea, giving rise first to methylurea and finally to urea. The possiblhty of ammonolysis of dimethylurea, occurring either alone or along with demethylation, eventually leading to urea, cannot be excluded at this stage. Paper chromatography of the raw urine from both rats and rabbits, as well as the concentrated effluent from the anionic and cationic exchange resins (neutral fraction) followed by the scanning of the radiochromatogram revealed significant peaks corresponding in Rf values to authentic samples of urea, methylurea and unchanged dmaethylurea as indicated m Figs. 1 and 2. Since the major peak, corresponding to methylurea, constitutes almost 67 ~o of the urinary radioactivity in both rat and the rabbit, the

considerable enrichment of the urinary urea fraction with 1'C could be due to some type of demethylation of dimethyhirea followed by methylurea ammonolysis and/or demethylation. This initial observation seems to suggest a stepwise demethylation of dLrnethyhirea. To obtain further evidence in support of demethylation of dimethylurea, it was decided to test the hypothesis of Mackenzie & du Vlgneaud (1948), according to whom the source of urinary urea carbon is the metabolic carbon dioxide in rats. The hypothesis was tested in rabbits using, besides N,N-dimethyl [14C-carbonyl]urea and other ~'C-labelled compounds, S-adenosyl [methyl-l'C]methionine. In order to test the above hypothesis, it was essential to determine the specific activities of the urinary urea and the respiratory carbon dioxide during the same interval of time in rabbits injected with 1'Clabelled compounds and to compute from this the ratio of the specific activity, urea/respiratory COz. The data showing the ratio of specific activmes of unnary urea and respiratory carbon dioxide with these ~'C-labelled compounds is shown in Table 2. Whereas the ratio of the specific actiwty of urinary urea and respiratory carbon dioxide is almost one in the case of p ' C methylamine hydrochloride and L-[methyl-~'C]meth] ionine treated rabbits, it is nearly 200 in the case of methyl[~'C-carbonyl] urea and 40 in the case of dlmethyl[~'C-carbonyl]urea treated animals. This significant increase in ratio, mainly due to a considerable increase in the specific activity of urinary urea as compared to respiratory carbon d~oxide, demonstrated the existence of a possible enzymatic stepwise demethylation that leads to the direct formation of p ' C ] urea from dimethyl[~4C-carbonyl]urea via methyl[x4C]urea. In fact, practically all the urinary radioactivity was confined to the so-called "neutral fractmn" which came through both the anionic and cationic exchange resins. The finding that danethylurea undergoes apparent stepwise demethylation is not without analogy as Geissbi~der et al. (1963) were first to demonstrate a progressive enzymatic N-demethylation of phenyl substituted dimethylureas used as herbicides. The

280

MOHAMMAD S DAR

same mechanism has been shown by Ernst & Bohme (1965) in rats and by Hodge et al. (1967) in dogs. Demethylatlon represents the major degradative pathway in the metabolism of these herbicides in plants and soils. The authors seemed to have overlooked the posslbdlty of ammonolysls as an alternatwe mechanism m the demethylation of substituted ureas The intermediary metabolltes of these urea herbicides, monomethyl and demethyl compounds, reported by Gelssbuhler et a l , could very well be the products of indirect demethylation wa ammonolysls The presence of methyl["C]urea as the major urinary metabohte of dlmethyl[14C-carbonyl]urea suggests the ease with which the first methyl group is removed The rate of &methylurea demethylatlon appears to be much faster than that of methylurea At present it Is difficult to explam thas significant difference m their rates of demethylation but it would be quite reasonable to link th~s to some specific properties and structural features of these compounds that so far have not been elucidated. However, in addmon to the above biochemical factors, the hydrolysis of p4C]urea due to bacterial ureases could also contribute to some extent towards the quantitative d~fferences between the urinary methyl[x'C]urea and [l'C]urea. Acknowledgements--This investigation was supported by funds provided by the Rockefeller Foundation, to the Department of Pharmacology through the Faculty o Science, Mahldol University, Bangkok, Thailand, and by a Pahlavl Umverslty Research Grant The author wishes to express his sincere thanks to Drs Herbert McKenms, J r , and E R Bowman for their interest and thoughtful assistance with the manuscript

REFERENCES ASATOORA M & SIM~NHOFFM L (1965) The origin of urinary dlmethylamme Bwchim. btophys Acta 111, 384-392 DAR M S (1970) Methylurea and methylamme--thetr metabolic interrelationships Va J Sci 21, 144 DAR M S (1970) Methylurea--ltS intermediary role in the physiological disposmon of methylamme Doctoral

Thesis Medical College of Virginia, Richmond, Va, USA DAR M S, BOWMANE. R & McKENNIS H JR (1969) The intermediary role of methylurea m the mammahan metabohsm of nicotine Fedn Proc Fedn Am Soc. exp Bwl 28, 545 ERNSTW & BOHMEC (1965) Uber den Stoffwechsel von Harnstoff-Herbiclden m Ratte I Mlttellung Monuron und Aresm Food Cosmetic Toxwol 3, 789-796 FOLIN O (1907) On the occurrences and formation of alkyl ureas and alkyl amines J Bwl Chem 3, 83-86 GEISSBUHLER H , HASELBACH C , AEBI H & EBNER L (1963) The fate of N'-(4-chlorophenoxy)-phenyl-N,Ndimethylurea (C-1983) in soils and plants III Breakdown in soils and plants Weed Res 3, 277-297 HODGEH C , DOWNSW L , PANNERB S, SMITHD W , MAYNARD E A , CLAYTONJ. W & RHODES R C (1967) Oral toxicity and metabolism of Diuron (N3,4-dichlorophenyl)-N,N-dlmethylurea in rats and dogs Food Cosmetic Toxtcol 5, 513-531 JEFFAY H. & ALVAREZ J (1961) Liqmd scintillation counting of carbon-14 Use of ethanolamme-ethylene glycol monomethylether-toluene Anal Chem 33, 612-615, MACKENZIE C G t~ DId VIGNEAUDV (1948) The source of urea carbon J Bwl. Chem 172, 353-354 PERRY T. L & SCHROEOERA W. (1963) The occurrence of amines m human urine Determination by combined ion exchange and paper chromatography J Chromatogr 12, 358-373. SALKOWSKIE (1877) Uber den Vorgang der Harnstoffblldung lm Thlerkorper und den Emfluss der Ammomaksalze aufdenselben Hoppe-Seyler' s Z Phystol Chem 1, 1-59 SCHIFrER J (1881) Uber der Schlksal des Sarkosms lm menschhchen Orgamsmus Hoppe-Seyler's Z Phystol Chem 5, 257-266 SCHMIDEBERG O (1878) Uber das Verhaltmss des Ammomaks und der prlmaren Monammbasen zur Harnstoffblldung lm Thmrkorper Arch exp Pathol Pharmakol 7, 1-14 STADTMANE R (1970) The Enzymes, 3rd Edn, Vol 1, p 401, Academic Press, New York WORTZ A (1851) Recherches sur les Ur6es Compos6es C r hebd sDanc. Acad S c i , Pans, 32, 414-419 Key Word Index--N,N-Dlmethylurea, demethylatlon; dlmethylurea metabohsm; demethylatlon of dtmethylurea.