JULIUS AXELROD Laboratory of Chemical Pharmacology, National Heart Institute, National Institutes of Health, U&led Stoles Public Health Service, Bethesda, Maryland Received for publication April 3, 1953 Numerous in vitro studies have implicated monoamine oxidase, phenol oxi- dase, and cytochromc osidnsc in the biotransformation of various sympatho- mimetic amines (Beyer, 1943). However, the part that these enzymes play in the intact animal is conjectural without definitive information concerning the chemical fate of the sympathomimetic amines in the body. The present communication, the first of a series of papers on the fate of sympathomimetic amines in the body, is concerned with the metabolic fate and physiological disposition of Z-ephedrine and its transformat,ion produck in a number of animal species. It will be shown that ephedrine is transformed along two metabolic pathways, one involving demethylation and the other hydrosy- lation to yield metabolic products which possess pressor activity and that various animal species show considerable differences in the relative importance of the two metabolic routes. CHEMICAL AIETIIVDS. Estimation of ephedrine and norephedrine: Both ephedrine and nor- ephedrine can be quantitatively extracted into benzene and assayed by the methyl orange reaction (Brodie and Udenfriend, 1945). The two drugs are determined in the presence of each other from calculations based on their partition ratios between petroleum ether and aqueous alkali. In the determination, one sample of biological material is extracted with benzene and the total methyl orange reacting material assayed. In another sample the dis- tribution ratio of the methyl orange reacting material between 1 volume of aqueous alkali and 3 volumes of petroleum ether is determined. The distribution ratio of the methyl orange reacting material in the unknown sample is a function of the relative concentrations of ephedrine and norephcdrine, since petroleum ether extracts about 90 per cent of the ephedrine and 30 per cent of the norephedrine. The concentration of ephedrine and nor- ephedrine iu the biological material is calculated by a simple algebraic equation described below. Procedure for plasma and urine: Pipet 15 ml. of plasma or diluted urine (containing 10 to 100 microgtn. of ephedrine plus norephedrine) into a 50 ml. graduated tube containing about 7 gm. of solid SaCl. Add 1 ml. of 10 N KaOH, dilute to 20 ml. with water and mix thoroughly (solution A). Total methyl orange reacting material (ephedrine plus norephedrinc) is determined as follows: Transfer 4 ml. of solution A to a 35 ml. glass-stoppered centrifuge tube containing 10 ml. of benzene2 and shake for 10 minutes. Centrifuge the tube and transfer 8 ml. of the benzene phase to a 15 ml. glass-stoppered centrifuge tube containing 0.5 ml. of isoamyl 1 Presented in part before the Amcricnn Society for Pharmacology and Experimental Therapeutics, Madison, Wisconsin, September, 1932. * Solvents arc purified by successive washings with 1 N NaOII, 1 N IICI, and 2 washings with water. 62 FATE OF l-EPIIEDIIINE c*3 alcohol.' Add 0.6 ml. of met,hyl orange reagent3 nnd shake vigorously for about 5 minutes. Centrifuge the tube and transfer 6 ml. of the supernatant benzene phnse to a cuvet con- taining 1 ml. of a solution of 2 per cent by volume of sulphuric acid in absolute ethanol. Work as rapidly as possible to minimize the adsorption of the methyl orange complex on the sides of the tube. Determine the optical density (T) in a spectrophotometer at MO mp. .4 reagent blank carried through the above procedure is used for the zero setting. The distribution ratio of the methyl orange reacting material between aqueous alkali and petroleum ether is determined as follows: Transfer 5 ml. of solution A to a 60 ml. glass- stoppered bottle containing 15 ml. of petroleum ether. Shake for 10 minutes and centrifuge the bottle. Transfer 4 ml. of the aqueous phase to a 35 ml. centrifuge tube containing 10 ml. of benzene, and determine the residual methyl orange reacting material (optical density D) as described above. Standard solutions of ephedrine and norephedrine are prepared by handling known amounts of each drug in the same manner as the unknown. Standards are run concurrently with the unknowns, since there is a small daily variation in the distribution ratios of ephed- rine and norephedrine betKeen aqueous alkali and petroleum ether. Optical densities of 0.220 and 0.190 are obtained (Coleman Model 6 epectrophotometer) when 10 microgm. of ephedrine and norephedrine, respectively, in 10 ml. of benzene react with methyl orange. Procedure for tissues: Emulsify about 10 gm. of tissue with 40 ml. of 0.1 N HCI in an electrically driven homogenizer consisting of a glass cylindrical cup in which a close fitting ground glass pestle is mechanically rotated.' Transfer 20 ml. of the tissue homogenate to a 50 ml. centrifuge tube and precipitate proteins by adding 10 ml. of a 20 per cent trichloro- acetic acid solution. Centrifuge the tube and transfer a 15 ml. aliquot of the supernatant solution to a 50 ml. centrifuge tube containing about 7 gm. of solid NaCl. Add 1.5 ml.>0 N NaOH, dilute to 20 ml. with water, and stir thoroughly. Proceed as described for plasma and urine for the estimation of ephedrine and norephedrine. Ephedrine and norephedrine added to biological material were recovered with adequate precision (95 f 5 per cent). Recoveries are less precise when the ratio of one compound to the other exceeds five. The ephedrine and norephedrine concentrations in the biological material are calcu- lated from the following equation: ED ND = E (optical density of ephedrine in the uukon-n solution) --- ET NT T = optical density of ephedrine plus norephedrine in the unknown solution D = optical density of ephedrine plus norephedrine in t,he unknown solution after shaking with petroleum ether ND - = distribution ratio of norephedrine between aqueous alkali and petroleum ether NT where Nn = optical density of standard norephedrine solution aft& shaking with petroleum ether and Nr = optical density of standard norephedrine solution ED - = distribution ratio of ephedrine between aqueous alkali and petroleum ether ET *A stock solution of methyl orange reagent is made by dissolving 509 mgm. of methyl orange in 100 ml. of warm water. The methyl orange solution is washed several times with equal volumes of chloroform. .4n aliquot of the resulting solution is diluted n-ith an equal volume of snturated boric acid solution immediately before use. 4 A glass homogenizer can be obtained from Scientific Glassware Co., Bloomfield, N. J. 64 where ED = optical dcusity of standard cphrdriue after shaking with petroleum ether aurl ET = optical density of standard ephedrine solution 7' - E = S (optical density of norephedrine in the unknown solution). EstituutiotE of p-hytfroryeyhcdrir~e plus p-hydrorynorephedrine: In the following pro- cedure p-hydrosyephedrine and p-hgdroxynorephedrine are not separated from each other but are isolated together from biological material by extraction into isoamyl alcohol at pH O-10. The extraction is augmented by saturating the aqueous phase with sodium chlo- ride. The phenols are returned to an aqueous phase by the addition of petroleum ether to the isoamyl alcohol solution and shaking with 0.1 N HCl. The aqueous solution is adjusted with SILOII to pH 9 to 10, aud reacted with 4-nminonntipyrine and potassium ferricyanide. The result,ing indophenols are assayed spect.rophotometrically at 500 rnp. Procedure: Pipet 5 ml. of biological material into a 60 ml. glass-stoppered bottle contain- ing 2 to 3 gm. solid SaCI. Adjust the pH to 9-10 by the addition of solid Na,C03. Add 25 ml. of isoamyl alcohol* and shake for 10 minutes. Centrifuge the bottle and transfer a 20 ml. of aliquot of the isoamyl alcohol to another 60 ml. bottle cont,aining 20 ml. petroleum ether and 5 ml. 0.1 N HCl. Shake for 5 minutes and transfer 4 ml. of the acid layer to a cuvet containing 1 ml. 1 A' SHIOH. (The pH should now be between 9-10.) Add 1 ml. 0.8 per cent potassium ferricyanide followed by 1 ml. of 0.25 per cent 4-aminosntipyrine, allow to stand for 5 minutes, and read the optical density at 500 rnp in a spectrophotometer. A blank containing 4-aminoantipyrine, potassium ferricyanide and NH&OH is used to set the spectrophotometer to read 100 per cent transmission. The distribution of p-hydroxyephedrine and p-hydroxynorephedrine in an isoamyl alcohol-salt saturated water syst.em is such that with volumes of 25 and 5 ml., respectively, about 90 per cent of the phenols are extracted in the organic phase. Standards are, there- fore, prepared by handling known amounts of either p-hydroxyephedrine or p-hydrosynor- ephedrine in the same manner as the u&non-n solution. p-Hydroxyephedrine and p-hy- droxynorephedrine added to biological material in amounts from 10 to 200 microgm. were recovered with satisfrictory precision (95 f 8 per cent). EXPEKIJWSTAL. Isolation and identification of ephedrine and norephedrine in the urine of dogs: Two dogs received 50 mgm. per kgm. of Z-ephedrine hydrochlo- ride int,raperitoneally and the urine was collected for the subsequent 24 hours. An aliquot of the pooled urine was made alkaline with XaOH and estracted with three volumes of chloroform. The extract contained considerable amounts of basic material Ivhich gave a methyl orange reaction (Brodie and Udenfriend, 1945). The chloroform extract was reduced to a small volume at room tempera- ture under a stream of nitrogen. The material reacting ivith methyl orange was subjected to a 99 transfer counter-current distribution, using equal volumes of pH 8.7 borate buffer (0.2 M) and chloroform. Under these conditions ephedrine haa a distribution ratio of about 1. After the counter-current distribution, the material reacting with methyl orange was determined in each tube. The total optical density of the methyl-orange react'ing material in each tube was plotted against the serial number of the tube. The distribution curve indicated that two compounds reacting with methyl orange were present (fig. 1). The contents of tubes 32-50, which contained material with partiCon ratios similar to that of ephedrilre, were pooled. The combined aqueous phase was made alkaline and the total basic material brought into the combined organic phase by shaking. The chloroform phase was evaporated to dryness at room temperature under a stream of nitrogen, and the residue was dissolved in anhydrous ether. Dry gnscous HCI was passed through the ether and the resulting prcripitate WLS recrystallized twice from an alcohol-ether mist.ure, yielding crystals whic~h meltcfl sharply at 220oC. (unwrr.). An authentic: sample of Z-ephedrine tlS.tlrochlfJritle melted at 219oC. (uncorr.) and a mixture of the two compounds showed no depression of the melting point. The ultraviolet absorption spectra of the ap- parent and authentic Z-ephedrine hydrochloride in 0.1 N HCl and 0.1 N XaOII were found to be almost superimposable. Conclusive evidence that the substance isolated from urine was Z-ephedrine was obtained by comparison of infra-red spectra of th"k isolated and authentic Z-ephedrine hydrochloride, which were identical. The material in tubes 58-72, isolated in the same manner as above, yielded a crystalline material with a melting point of 175176oC (uncorr.). An authentic sample of Z-norephedrine hydrochloride melted at 175oC. (uncorr.) and a mixed TUBE NUMBER FIG. I. Counter-current distribution of methyl orange reacting material isolated from the urine of two dogs that received 50 mgm. per kgm. of Z-ephedrine hydrochloride. System 0.2 M borate buffer pH 8.7, and chloroform (equal volumes). meiting point of the two compounds showed no depression. The ultraviolet absorption spectra of the isolated metabolite and of authentic I-norepheclrink in 1 N HCl and 0.1 N NaOH were found to be almost superimposable. Apparent Z-norephedrine hydrochloride isolated from the urine gave the following per cent composition on analysis: Calculated: for C~H1~OXCl: C, 57.59; H, 7.52 Found : C, 57.25; H, 7.41 Conclusive evidence bhat the substance isolated from the urine mas Z-norephed- rine was obtained by qomparison of infra-red spectra of the isolated and authen- tic I-norephedrinc hydrochloride, which were identical. Isolatiorb and identiJication of p-hydrosyephedrine ad p-hydrorynorephedrirle in the urine oftlogs: An aliquot of t,he poolecl urine was saturated with solid sodium chloride, adjusted to pTI O-10 with solid Sa2C03, and extracted three times with 66 JULIUS ASELWD equ31 voliu~~es of isonmyl alcohol. The isoamyl extract was reduced to a 15 ml. volume in ZWUO. I'hcnolic material was returned t.o an aqueous phase by adding two volumes of petroleum ether to the isoamyl alcohol cst,ract and shaking with 10 ml. of 0.1 A' IICI. The acid extract was adjusted to pEI 10.3 with solid Ka&PO, and NaOII (1 N). The phenolic material was subjected to a 21 trans- fer counter-current distribut.ion, using equal volumes of ~1-1 10.3 borate buffer (0.2 nf), and isoamyl alcohol. Under these conditions the phenolic material, as estimated by the 4-aminonnt.ipyrine react,ion (see Chemical Methods) was dis- tributed about equally between each phase. After the counter-current distribu- 20 - I I I I - ,/ I 0 5 10 15 20 25 TUS& NLMER FIG. 2. Counter-current distribution of phenolic compounds isolated from the urine of two dogs that received 50 mgm. per kgm. of Z-ephedrine hydrochloride. System 0.2 M borate buffer pH 10.3, isoamyl alcohol (equal vohm~cs). Experimental distribution (solid line), theoretical distribution for p-hydroxynorephedrine (dashed line), experimental distribution minus theoretical distribution (dotted line). tion, the phenolic material was determined in each tube. The total optical density of the phenolic material in each tube was plotted against the serial number of the tube. The distribution curve showed the presence of more than one phenolic product (fig. 2). Tubes 4-10 contained material with partition coefficients similar to that of authent,ic p-hydroxynorephedrine. The theoretical distribution curve of p-hydrosynorephedrine was calculated from its experimentally established partition ratio. Subtracting the calculated curve from the observed curve showed the presence of relatively small amounts of a second phenolic compound (tubes 1 l-20). The material in tubes 15-19 had approximately the same partition ratio as that of p-hydroxyephedrine. Further evidence for the identity of the phenols was obtained by the technic of comparative distribution ratios (Brodie ct a~., 1947). Distrihut.ion rat.ios of the phenolic compounds in tubes 4-10 (apparerlt p-hydrosynorcphedI.irle) and tubes 15-19 (apparent ?I-hydrosyeptlctlrillc) be- tween organic solvents and buffers at various pII values were compared \vith those of authentic samples (table 1). The results confirmed the presence of p-hydrosynorephedrine and p-hydrosyephedrine. The urine was also examined for the presence of 3,4-dihydrosynorephedrine, a potent pressor agent, by the method of Beyer and Shapiro (1945). No detect- able amounts'd;f this compound were found. The amounts of ephedrine and its transformation producb excreted in urine of dogs: The urine of five dog-:, each of which had received 50 mgm. per kgm. of l-ephedrine hydrochloride intraperitoneally was analyzed for ephedrine and its transforma.tion products. The urine had been collected for 24 hours following t.he drug administration; negligible amounts of ephedrine or its transformation TABLE 1 The distribution of apparent hydroxynorephcdrine and hydrozyephedrine and the aufhenfic substances between organic solvents and waler at various pH values The fraction-of the compounds extracted at various pH values is erpressed as the ratio of the amount of compound in the organic phase to total compound. PH _--___ 6.0 7.4 a.2 9.6 10.3 11.4 10.3 soL"LhT Isoamyl Alcohol Isoamyl Alcohol Isoamyl Alcohol Isoamyl Alcohol Isoamyl Alcohol Isoamyl Alcohol Ether 0.1s 0.34 0.75 0.80 0.72 0.26 0.08 1 - - 0.18 0.35 0.75 0.81 0.73 0.27 0.10 products were excreted `after this time. About 6 per cent of the administered ephedrine was excreted as the unchanged drug, about 58 per cent as norephedrine, and about 1.5 per cent as p-hydrosyephedrine plus p-hydrosynorephedrine (table 2). Plasma levels of ephedrine and norephedrine ajtw the administration of ephedrine to dogs5: Plasma concentrations of ephedrine and norephedrine were measured at various time intervals following the intravenous administratiou of 2d mgm. per kgm. of Z-ephedrine hydrochloride (fig. 3, typical of four esperiments). Plasma level,3 of ephedrine deeliked about 50 per cent per hour, whereas those of its derived I;roduct, norephedrine, rose to considerable levels, indirat.ing that, ephedrine was rapidly demethylated. The levels of norephedrine persisted long beyond the time when those of ephedrine had declined to negligible proportions. The distribution of ephedrine and norephedt-ble in tissrces: The extent to which `Ephedrine, and norephedrine values in this rebort are espreased in terms of the free base. Gs JCLICS ASE:I,IfOD ephedrine and ~~orcphcclrine Gere bound to plasma proteins was examined by dialysis against isotonic phosphate buffer of pII 7.4 at 3i"C. for 20 hours. Vi&- ing mcmbrnnrs UTW used as dialysis bugs. At plasma concentrations of 10 mg;n. per liter :lpprusim:itcly 1'2 per cent of cpl&rine and 20 per cent of norcphedrine were found to be bound to the nondiffusible constituents of the proteins. TABLE 2 The metabolic fafe of ephedrine in the dog Rc~overy of ephedrine nnd its metabolic products from the urine of dog given5Omgm./kgm. of l-ephedrine intrnperitoneally The urine was collected over a period of 24 hours. The proportions of the various me- tnbolites in the urine are espressed in percentage of the amount of ephedrine administered. Doe NO. EPIIEDPlh-E % 1 4.2 2 2.6 3 2.1 4 4.1 5 20.0 - I------ % % 58 1.2 65 1.6 50 1.6 63 1.9 53 - TIME IN HOURS FIG. 3. Plasma levels of ephedrine (solid line) and norephcdrine (dotted line) after the intravenous administration of 20 mgm. per kgm. of Z-ephedrine hydrochloride to a dog. The di:;t.ribution of ephedrine and norephedrine was examined in representa- tive tissues of two dogs which were given 50 mgm. per kgm. of Z-ephedrine hydrochloride intraperitoneally. The animals were sacrificed by an intravenous `injection of air one and two hours after the administration of the drug and the tissues sampled immediately afterwards. Io dog 6, sacrificed one hour following drug adn-Gstration, the tissue concentrations of ephedrine were higher than those of norephedrine (table 3). Both compounds wc~t lwalized in organ tis~l1c.s to a considerable estent wit.h ncgligiI)le loc*alization in body fat. `l'he high cou- ceutratiou of the compounds iu the l,railr and cercbro~pinal fluid would suggr:,$t that there is little hindrance to their passage through the blood-braiu barrier. The high concentration of ephedrine in the cerebrospinal fluid as compared to plasma is unusual and may be due to the relatively slow diffusion of the drug from cerebrospinal fluid as the drug rapidly disappears from plasma. In dog 7, sacrificed 2 hours following the administration of the drug, the concentration of norephedrine M-tissues is considerably higher than the concentration of ephedrine indicating that most of the ephedrine had been demethylated within two hours. TABLE 3 Distribution of ephedrine and norephedrine ajter fhe administration of ephedrine Two dogs received 50 mgm./kgm. I-ephedrine intraperitoneally. Dog 6 was sacrificed one hour and bog 7 was sacrificed two hours after the drug administration. no06 DOG7 Ephedrine Norephedrine Ephedrine mgm.figm. wdkgm. mgm./kgm. hz;p.h;d$ Plasma ............................. 8.6 4.6 0.9 5.7 CSF ................................ 22 0 4.0 4.1 Liver ............................... 172 31 2.5 41 Lung ............................... 85 21 16 44 Kidney ............................. 108 8.5 28 77 Brain ............................... 127 5.5 18 29 hluscle ............................. 72 5.0 8.3 12 Heart .............................. 68 20 8.3 15 Spleen .............................. 103 9.3 15 40 Fat. ................................ 6.8 0 11 4.4 Fate of the melaboliks of ephedrine in the dog: The importance of norephedrine, an active pressor agent (Chen, Wu and Hendrickson, 1929), in the overall metabolic transformation of ephedrine, prompted a study of the fate of this compound in the dog. Two dogs (1 and 2) were given 20 mgm. per kgm. of I- norephedrine hydrochloride intravenously and the plasma levels and urinary excretion of the drug were measured. Plasma levels of norephedrine declined at a rate of about 25 per cent per hour (fig. 4), as compared with ephedrine which disappeared at a rate of 50 per cent per hour (fig. 3). About 72 per cent of the administered norephedrine was escreted unchanged, the remainder disappearing by an unknown route. * Only small amounts of hydrosylated ephedrine and norephedrine, bot.h potent pressor agents (Chen, Wu and Hendrickson, 1939), were found in the urine but the possibility remained that these compounds may have been formed in con- siderable amounts and then further met.abolized. To investigate this possibility, the fate of p-hydrosyephedrine and p-hydrosynorephedrine was examined in a dog. Dog 2 was given 15 mgm. per kgm. of dl-p-hydrosyephedrine hydrochlo- ride intravenously, following which plasma levels of the drug and the urinary escretion of the free n11t1 conjugated compound were measured at, various illtcr- vals of time. The wmpound UXJ found to clisappenr from the plasma at a rate of -15 per cent per h.~Ul . :\lJOUt GO per cellt of the :tt~n~inistcrcd drug \vas foulId in the uriIlc as free and conjug;lted p-hS'drus3.cphetlrille. IWO\\-ing the administration of dE-p-hydrosynorephedrillc in the same dosage, the pl:wi~n levels declined about -10 per cent per hour. About 50 per cent of .the administered drug was recovered in the urine as free and conjugated p-hydrosy- norephedrine These results indicate that, both p-hydrosgephedrine and p- h~clrosyI~orcpheclri~~e are relatively stable in the body. It may be concluded from these observations that hydrosylation is a relat,ively minor pathway in the metabolism of I-ephedrine in the dog. I I I I I I I I I 1 2 3 4 5 6 7 8 TIME IN HOURS FIG. 4. Plasma levels of norephedrine after the intravenous administration of 20 mgm. per kgm. of Z-norephedrine hydrochloride to a dog. Fate of ephedrine in gu.inea pigs, ruts and rabbits: The metabolic transformation of ephedrine was studied in guinea pigs, rats (Sprague-Danlcy), and rabbits. Each animal was given 50 mgm. per kgm. of Z-ephedrine hydrochloride intra- peritoneally and the subsequent 24 hour urine sample was esamined for ephedrine and metabolic products (table 4). Dogs and guinea pigs exweted only small amounts of ephedrine, considerable amounts of norephcdrine, and a small amount of the hydrosylated derivatives. Rats, on the other hand, excreted relatively small amoulits of norephedrine but considerable quantities of both ephedrine and its hydrosylated derivatives. These results suggest that, the dc- methylation process is relatively slowx in the rat so that hydrosylation and rertal elimination of ephedrine arc favored. The rabbit escret,ed only small amounts of ephcdrilw, norephedrine, and p-hydrosyephedriue. To further dclincnte the intermediate metabolism of ephcdrinc in the rabbit, the fates of norcphctlrillc and p-hydrosycphedr.i~Ic were ernmirtctl itI thi.3 ypc:pips. After the administ~nrt inn of 10 mgm. per kgm. of dl-p-h-drc,septlcrlt.irIc, to 2 rshhits, the mijor portiwl, Rhlt 65 per cent of the atlmitli5iterd WI~~OLLIII~, was escreted in the free and cwjugated form. R'hen 25 mgm. per kgm. of 1- norephcdrine was administered to `2 rabbits only 3 per cent of the drug \vas escretcd. These results suggest that in the rabbit ephedrine is demethylated to norephedrine which in turn is extensively metabolized. TABLE 4 Fate of ephedrine in a number of species Fifty mgm./kgm. Z-ephedrine were administered intraperitonexlly to c:rc!l animal. The urine was collected over a period of 24 hours. The proportion of the various me- tabolites is expressed in percentage of the amount of eDhedrine administered. - Dog ................................ Guinea Pig.....: ................... Rat ........ ............ . ........... Rabbit .............................. % % % 5 6.5 57.8 1.5 3 2.0 3s.5 0.9 4 32.0 7.5 12.8 4 0.1 1.8 I.9 DISCUSSION. The following scheme for the route of metabolism of Z-ephedrine in the dog is suggest,ed by the studies described in this report. OH OH OH GLCH I l-1 HI 3 6 C-C-CH, NHCH3 -+ iI AH 2 OH p-hydroxyephedrine l-ephedrine I-noI:ephedrine / OH p-hydrosynorcphcdrine The main route of metabolism involves rapid demcthylation to Z-norcphtdrine, a relatively stable and potent pressor agent. It may be concluded therefore that the activity of ephedrine is mediated to a considerable estent through norcphed- rine. Other, though minor, routes of metnbo!ism involve hydrosyhltion of both ephcdri!le and norephcdrine to yield t,he corresponding p-hydrosy deri~xtives, both of whit+ are also potelit pressor ngcnts. `X0 cvidcncc was found for the formai ion of 3 , l-dih~clros~cphc~ll~i~~c or 3, -~-clihyd~~os~t~urc~~~llctlrir~c. Sorcphctlrinc is rclati\.ely stalk iI1 the clog and is escxted in the urillc mainly ullc~h~mgf~d. f?:~hlhti~Jl~s of rci~al excretion of iiorepllctlrinc from plasma levels and urinary escretion of the drug yielded values which NXYC considerably higher than the glomcrular filtration rate, suggesting that a secretory transport mechn- nism is involved in its escretion. Richter (1939) reported that in mm ephedrine is excreted in the urine almost elltircly unchanged and ascribed the stability of the drug ilt aiuo to the failure of monoamine osidase to catalyse the deamination of this compound. However, the procedure used by Richter for the estimat,ion of ephedrine would not have distinguished bet\\-een ephedrine and norephedrine. Previous work has shown t,hat a number of alkylamines, including aminopyrinc (Brodie and Aselrod, 1950), meperidine (Lief et al., 1952) and mephobarbital (Butler, 1953), are demethylated in man; it seems likely that the same would hold true for ephed- rine. However, at present it cannot be said whether the results obtained with dogs using large doses of ephedrine would carry over into man employing thera- peutic doses of the drug. Studies on the fate of Z-ephedrine in man are planned for a future investigation. There are considerable differences in the metabolism of ephedrine by different species. In the dog and the guinea pig demethylation of the drug proceeds rapidly const,ituting a major route of biotransformation. The rat, on the other hand, demethylates ephedrine slowly and considerable amounts of the clrug are excreted both unchanged and as hydrosylated derivntives. The rabbit excretes only negligible &nounts of norephedrine but demethylation cannot. be ruled out since norephedrirle is almost completely metabolized in this animal. Methods for the estimation of ephedrine and its metabolic products, noreph- edrine, p-hydrosynorephedrine and p-hydrosyephedrine, in biological materials, arc described. In the dog the main route,of biotransformation of ephedrine involves demethy- lation to norephedrine. The rate of demethylation is rapid, indicat,ing that the activity of ephedrine is mediated largely through norephedrinc. Korephedrine is excreted in the urine mainly unchanged. A minor route of metabolism of ephedrine involves hydrosylat,ion of the aromatic nucleus to form p-hydrosy- ephedrine and p-hydrosynorephedrine, which arc excreteel partly free and partly as conjugates. Roth ephedrine and norephedrine are highly localized in various organ tissues. Dcmethylation to norephedri%e is a major route of metabolism in the dog and guinea pig and ti minor pathway in the rat. On the other hand, hydroxylation of the drug constitutes a minor pathway of metabolism in the dog and guinea pig but a major one in the rat. AC~SO~~.LEDGXIEST. T~I: author wishes to tkank Drs. I<. H. Beyer, I<. I<. Cherr, and G. E. Ullyot for kindly supplying I-norcphcdrine, dl-3,4-dihydrosy- fyhetli*il~c, arid dl-p-lIS.tlrosyrIo~cpllc:tl~irIE, and 1)r. B. U. IQdie for help in prqmydion of this Insnuscript. ILEFJ~~:I~~SCXS BEYER, K. II.: Physiol. Rev., 26: 169,19-1G. BEYE~, I<. II., AND SHANR~, S. II.: Am. J. Physiol., 144: 321, 1925. BRODIE, B. B., AND AXEI,R~D, J.: THIS JOURNAI,, 99: 171, 1950. BRODIE, R. B., ASD UDENFRIEND, S.: J. Biol. Chem., 168: 705, 1945. BRODIE, B. B., UDEXFRIEND, S., AND BAER, J. J.: J. Biol. Chem., 168: 299,19-17. BUTIZR, T. C.: TIIIS JOURNAL, 106: 235, 1952. CEIEX, K. I<., Wu, C. K., AND HEXDRICKSOX, W.: THIS JOURNAL, 36: 3G3, 1929. LIEF, P. A., BURNS, J. J., PAPI'ER, E. M., BERQER, B. L., WOLLACK, A., AND BRODIE, B. I%. Fed. Proc., 369: 11, 1952. RICHTER, D.: Biochem. J., 32: 1763,1938.