THE FATE OF PROCAINE IN MAN FOLLOWING ITS INTRAVENOUS ADMINISTRATION AND METHODS FOR THE ESTIMATION OF PROCAINE AND DIETHYLAM1NOETHANOL

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1 THE FATE OF PROCAINE IN MAN FOLLOWING ITS INTRAVENOUS ADMINISTRATION AND METHODS FOR THE ESTIMATION OF PROCAINE AND DIETHYLAM1NOETHANOL BERNARD B. BRODIE, PHILIP A. LIEF AND RAYMOND POET Research Service, Third (New York University) Medical Division, Goldwater MemoriaL Hospital, and the Department8 of Biochemistry and Anesthesia, New York Univer8ity College of Medicine, New York, N. Y. Received for publication August 11, 1948 The local anesthetic, procaine, has been administered intravenously for the control of pain associated with burns, fractures, etc., and in the post-oative iod following the trauma of surgical procedures (1, 2, 3). It has also been used for disorders of cardiac rhythm observed during anesthesia (4). Studies were undertaken to identify the transformation products of procaine in the body, and to study the extent to which the activity of the drug is limited by its transformation, or is associated with the derived products. This pa deals with the physiological disposition of procaine and the identification of its derived products. It has been demonstrated that an enzyme exists in plasma which catalyzes the hydrolysis of the drug to p-aminobenzoic acid and presumably, diethylaminoethanol (5). Little positive information is available, however, concerning the fate of procaine in vivo. This is due in no small part to the lack of a suitable method for the determination of procaine and its transformation products in biological tissues. CHEMICAL METHODS. A study of the metabolic fate of procaine required sensitive metlods for the estimation of procaine and its metabolic products in biological material. Determination of Procaine. Methods for the estimation of procaine in biological fluids have been previously described. The chief difficulty involved in its estimation is the separation of procaine from its hydrolytic product, p-aminobenzoic acid. The method of Kisch and Strauss separates procaine from p-aminobenzoic acid by an extraction procedure, and then estimates it by the Bratton and Marshall reaction (6). This procedure includes no provision for the inhibition of the enzymatic hydrolysis of the procaine which occurs after the blood is drawn. Moreover, the method is not sensitive enough to measure the small amounts of procaine which occur in plasma during its intravenous administration to man. The method described by Graubard is unsuitable since the measurement includes the large excess of p-aminobenzoic acid derived from the hydrolysis of procaine in the body (7). The method described below is a modification of that of Kisch and Strauss. The in vitro hydrolysis of the procaine is prevented by the addition of sodium arsenite to the blood. The sensitivity of the measurement is considerably enhanced by forming the azo dye in a small volume and assaying its concentration by microspectrophotometry. This mits the estimation of plasma procaine concentrations as low as micrograms liter. The procaine is isolated from the biological material by extraction into ethylene di- 1 This work was supported by a grant from the Institute for the Study of Analgesic and Sedative Drugs. The procaine (novocaine) used in these studies was obtained through the courtesy of the Department of Medical Research, Winthrop-Stearns Inc. 359

2 360 B. B. BRODIE, P. A. LIEF AND R. POET chloride, returned to dilute acid, and then diazotized and coupled with N (1-naphthyl) ethylene diamine. The resulting dye is assayed in a spectrophotometer adapted to small volumes (8). The specificity of the method was appraised by a distribution technique (9, 10). The results obtained indicate that the substance measured in biological material is identical with authentic procaine. Preparation of plasma samples : Human plasma contains an enzyme which markedly accelerates the hydrolysis of procaine to p-aminobenzoic acid and diethylaminoethanol (5). Procaine in human plasma completely disappears in about two minutes unless the enzy. matic activity is inhibited (table 5). The blood is therefore rapidly drawn and immediately placed in a test tube containing sodium arsenite (2 drops of 50 cent solution cc. of blood). The contents are mixed by inversion of the tube which has been covered by a microscope slide. The plasma is separated from the cells and analyzed within an hour. If it is inconvenient to analyze the sample within this time, the plasma proteins may be precipitated with trichioracetic acid and the procaine estimated in the filtrate. The procaine is stable for at least several days in the filtrate. Aliquots are neutralized with sodium carbonate, buffered to ph 9 and the procaine estimated as described below. Procedure. Add 1 to 5 cc. of plasma or suitably diluted urine (containing up to 5 micrograms of procaine) and 1 cc. of 0.8M borate buffer (ph 9) to 20 cc. of ethylene dichloride in a 60 cc. glass-stoped bottle. Shake for 10 minutes. Transfer the contents of the bottle to a test tube and centrifuge for 5 minutes. Remove the aqueous phase by aspiration. Transfer 15 cc. of the solvent phase to a 40 cc. conical glass-stoped tube containing 1 cc. of in HC1. Shake for 5 minutes and then centrifuge. Introduce a finely drawn glass tube to the bottom of the lower solvent phase and gently aspirate all but the last traces of the solvent. Add 0.05 cc. of 0.1 cent sodium nitrite solution. Wait 5 minutes, add 0.05 cc. of 0.5 cent ammonium sulfamate solution3. After 3 minutes add 0.05 cc. of 0.1 cent N(1-naphthyl) ethylene diamine dihydrochloride. Allow 20 minutes for the color to develop fully. Determine the optical density of the dye at 550 mt in the Coleman Model 6 spectrophotometer adapted to microspectrophotometry (8). A reagent blank is run through the procedure. This reagent blank should not give an optical density of more than when in HC1 to which the diazotizing and coupling reagents have been added is used for the zero setting. Standards are prepared by taking 1 cc. of standard solution and adding nitrite, sulfamate and coupling reagent as described above. A blank of in HC1 to which the above reagents have been added is used for the zero setting. The optical densities were found to be proportional to concentration. An optical density of about was obtained in the adapted Coleman Model 6, when 1 microgram of procaine was run through the procedure. Procaine added to plasma and urine in amounts from 0.5 to 3 micrograms was recovered with adequate precision (98 ± 6 cent). Determination of Diethylaminoethanot. A reaction previously described (9), for the estimation of basic organic compounds was used in the estimation of diethylaminoethanol. This reaction involves the formation of a salt of the organic base with methyl orange. This salt is extracted into ethylene dichloride and estimated directly in the solvent. Difficulties were encountered in the estimation of diethylaminoethanol by this method because of the marked water solubility of its methyl orange salt. This solubility was greatly reduced by A technical grade of ethylene dichloride is purified by successive washings with in NaOH, in HC1 and three washings with water. 1.5 cent by volume of isoamyl alcohol, reagent grade, which has been similarly treated, is added to the solvent to minimize the adsorption of the compound from the solvent onto the glass surface. A non-procaine blank, which does not react with the coupling reagent, is extracted from urine. This blank is corrected for by reading the optical density of the solution at this stage before the addition of the coupling reagent and subtracting the reading from the final optical density of the dye.

3 PROCAINE IN MAN 361 increasing the concentration of methyl orange, and by reducing the volume of the aqueous methyl orange phase. The diethylaminoethanol is isolated from biological material by extraction into ethylene dichloride. The ethylene dichloride phase is shaken with methyl orange at ph 5 and the excess methyl orange is removed. The methyl orange, which dissolves in the solvent through salt formation in amounts equivalent to the contained base, is estimated spectrophotometrically. The specificity of the method was appraised by the distribution technique. The results obtained indicate that the substance measured in the biological material is identical with authentic diethylaminoethanol. Procedure. Add 2 cc. of plasma or suitably diluted sample4 (containing up to 30 micro. grams of diethylaminoethanol), 1 cc. of in NaOH and 3 grams of potassium iodide5 to is cc. of ethylene dichioride in a 60 cc. glass-stoped bottle. Shake for 10 minutes. Transfer the contents of the bottle to a test tube and centrifuge for 5 minutes. Transfer as much of the sunatant solvent phase as possible to a 60 cc. glass-stoped bottle containing 0.1 cc. of methyl orange reagent. Shake for 5 minutes, transfer the contents to a test tube and centrifuge at high speed. Completely remove the sunatant methyl orange layer by aspiration. Pipette 10 cc. of the solvent phase into a colorimeter tube containing 2 cc. of a solution of 2 cent by volume of sulfuric acid in absolute alcohol. Determine the optical density in a spectrophotometer at 540 m. A reagent blank is run through the same procedure. The reagent blank should not yield an optical density greater than when ethylene dichioride plus the alcoholic sulfuric acid is used for zero setting. (Coleman Model 6 spectrophotometer.) Standards are prepared by taking 2 cc. of standard solution, adding 1 cc. of in NaOH, 3 grams of potassium iodide and 15 cc. of ethylene dichioride and handling in the same manner as the unknowns. An optical density of about 0. is obtained when 5 micrograms )f diethylaminoethanol are run through the procedure. Diethylaminoethanol added to plasma and urine in amounts from 5 to 30 micrograms was recovered with satisfactory precision (105 h 5 cent). Procedure for total p-aminobenzoic acid (Jree and conjugated). The conjugated forms of p-aminobenzoic acid are deacetylated to p-aminobenzoic and p-amninohippuric acids. The latter compounds are diazotized and coupled with N(i-naphthyl) ethylene diamine. The resulting dyes are assayed at 5.50 m. The dyes formed from both p-aminobenzoic acid Organ tissues are prepared for analysis by emulsification in acid as described in a previous pa (10). The addition of potassium iodide to the aqueous phase makes it heavier than the ethylene dichloride phase. This not only facilitates the handling of the solvent phase, but also decreases the blank to some extent. #{149} The methyl orange reagent used previously in the estimation of organic bases was a saturated solution in 0.5M boric acid and contained about 90 mgm. of methyl orange cc. (9). This solution is too dilute to give a suitable reaction with diethylaminoethanol. However methyl orange is about seven times more soluble in ordinary water. A saturated solution of methyl orange in water when added to a boric acid solution results in a susaturated solution of methyl orange from which the latter does not crystallize out for some time. Advantage of this phenomenon is taken to produce a relatively high concentration of methyl orange in boric acid solution. A saturated aqueous solution of the sodium salt of methyl orange is washed several times by shaking with half its volume of ethylene dichloride. The methyl orange reagent is made by diluting this solution with an equal volume of saturated boric acid solution. This dilution is made just prior to using since the methyl orange precipitates out within an hour. The diethylaminoethanol estimated in plasma includes that derived from the in vitro hydrolysis of the procaine also present. The concentration of procaine is usually so small as to involve a negligible correction.

4 362 B. B. BRODIE, P. A. LIEF AND B. POET and p-aminohippuric acid yield the same optical density mole. The estimation may therefore be expressed in terms of total p-aminobenzoic acid. The procaine present is also included in the estimation but is so small that it may be neglected in the calculation of total p-aminobenzoic acid. The total p-aminobenzoic acid is determined in a filtrate of plasma. To deproteinize the plasma, add 5 cc. of 50 cent trichioracetic acid to 2 cc. of plasma and 3 cc. of water. Transfer 5 cc. of the plasma filtrate or diluted urine to a test tube containing 1 cc. of 12N HC1. Heat on the water bath for 1 hour. Estimate the resulting aromatic amines by the Bratton and Marshall reaction (11). Evidence for the identity of the substances determined in biological fluid. Conclusions concerning the fate of procaine depend upon a knowledge of the identity of the substances in the biological material. A technique, previously described by us (9, 10), mits the identification and, to a considerable degree, the establishment of purity of a substance being TABLE 1 Distribution of procaine and apparent procaine between ethylene dichioride and water at various ph values The apparent procaine was obtained by extraction with ethylene dichloride of the plasma of a subject receiving procaine by intravenous infusion. The compound was returned to dilute acid. Aliquots of this solution and an authentic procaine solution were adjusted to various ph values and shaken with equal volumes of ethylene dichloride. The fraction of the compounds extracted at various ph values is expressed as the ratio of the amount of compound in the organic phase to total compound. ph ATEZNTIC PZOCAIN measured. It involves a comparison of the distributions of the substance with those of the authentic substance in a two-phase system consisting of an organic solvent and water at various ph values. Dissimilar distributions indicate the presence of a substance different from the authentic compound. To escape detection, a transformation product would have to have not only a similar dissociation constant but identical solubility characteristics in two solvents. The examination in the case of procaine and diethylaminoethanol was made with ethylene dichloride extracts of plasma from patients who had received procaine intravenously. The partitions of procaine and diethylaminoethanol between ethylene dichloride and water at various ph values was compared with those of the apparent compounds from the biological fluids. The results with each substance showed that, within eximental error, the apparent and the authentic compound had the same solubility characteristics and were therefore presumably the same compound (table 1-2). FATE o PROCAINE IN MAN. Procaine and its transformation products found in urine after procaine administration. Information concerning the metabolic products of procaine was obtained from the urine of 5 subjects given 2 grams of the drug by intravenous infusion. The infusion time varied from 45 to 125 minutes. The urines were collected over a iod of 24 hours. The excretion of the various transformation products subsequent to this time was negligible. Approximately 2 cent of the administered procaine was excreted unchanged, indicating that the drug is almost completely metabolized in the body. p.-

5 PROCAINE IN MAN 363 Aminobenzoic acid and itsvarious conjugates found in the urine8 were equivalent to about 80 cent, while the diethylaminoethanol was equivalent to about 30 cent of the administered procaine (table 3). Fate of diethylaminoethanol and p-aminobenzoic acid in the body. Further information concerning the fate of procaine was obtained by studying the fate of its metabolites. A combined dose of 1 gram of diethylaminoethanol and 1 TABLE 2 Distribution of diethylaminoethanol and apparent diethylaminoethanol between ethylene dichioride and water at various ph values The apparent diethylaminoethanol was obtained by extraction with ethylene dichloride of the plasma of a subject receiving procaine. Aliquots of this solution and of an ethylene dichloride solution of authentic diethylaminoethanol were shaken with j volume of water at various ph values. The fraction of the compound extracted at various ph values is expressed as the ratio of the compound in the organic phase to total compound. H AUTifENTIC DIETEYLAMINOETHANOL TABLE 3 The metabolic fate of procaine in man APPAZENT DIETRYLAMINOETSANOL 720M PLASMA Recovery of procaine and its metabolic products from the urine of subjects after the intravenous administration of 2 grams of procaine. The urine was collected over a iod of 24 hours. The proportion of the various metabolites is expressed in centage of the theoretical amounts that could occur from the amount of procaine administered. PIOCAINE ceisi DIZTEYLAMINOETEANOL er ce,ji P.AuINoBzNzoIc ACID TREE AND CONJUGATED ceut gram of p-aminobenzoic acid was administered intravenously in 250 cc. volume to 2 subjects. Urine was collected over a iod of 24 hours. About 33 cent of the administered dose of diethylaminoethanol was excreted unchanged. About 90 cent of the p-aminobenzoic acid was recovered in the urine in the form of p-aminobenzoic acid and its various conjugates. 8 The identity of the various p-aminobenzoic acid conjugates was not determined since this information was not relevant to the present problem. These conjugates have been reputed to be predominantly free and acetylated p-aminobenzoic acid.

6 364 B. B. BRODIE, P. A. LIEF AND R. POET The above results indicate that about the same fraction of p-aminobenzoic acid and diethylaminoethanol are excreted whether they are given directly or combined as procaine. This suggests that the main, if not the only, route of TABLE 4 Plasma levels of procaine and its metabolites during and after the intravenous infusion of grams to man TIME PROCAINE DIETEYLAMINOETUANOL ACIDTOTAL TREE PA1tINOBENZOIC AND CONJUGATED - isjsstes after start of isfusios i*wes after end of infusson muwles after start of infusion jsinuiis after end of infusion 20,*g*s./L Subject Subject A B *g*./l snge../l. metabolism of procaine is hydrolytic cleavage to p-aminobenzoic acid and diethylaminoethanol as follows: N11/J9C00CH2CH2N H6 of 45 to 125 minutes. The concentrations of procaine in plasma were consistently low during the infusion (table 4-2 typical eximents). The diethylamino- - NH2()000H + HOCH2CH2N The diethylaminoethanol is further metabolized but the nature of this transformation is not yet known. Procaine and its transformation products found in plasma. Two grams of procaine were administered to 5 subjects by intravenous infusion over iods 02118

7 PROCAINE IN MAN 365 ethanol and total p-aminobenzoic acid levels on the other hand were relatively high and rose steadily during the time of the infusion. At the termination of the infusion, the plasma levels of procaine soon were negligible, whereas those of diethylaminoethanol and total p-aminobenzoic acid sisted for some time. These results indicate that procaine, after its intravenous injection, is hydrolyzed in the body at an unusually rapid rate. The role of plasma in the breakdown of procaine. The extent to which the enzyme in plasma is responsible for the breakdown of procaine was investigated. Procaine was added to fresh human plasma at 37#{176}C to a concentration of 5 micrograms cc. Samples for procaine analysis were taken in 1, 2, 3, and 6 minutes. It is evident from the results that procaine was hydrolyzed extremely rapidly in plasma in vitro (table 5). Eighty to 103 cent of the procaine broke down within 2 minutes (4 eximents). The derived products were identified as p-aminobenzoic acid and diethylaminoethanol by means of the distribution technique as described under methods. - TABLE 5 The in vitro hydrolysis of procaine in human plasma at 37#{176}C. One-tenth cc. of 0.02 cent procaine solution was added to 4cc. of plasma. The extent of hydrolysis is expressed in centage of the amount of procaine added to the plasma. TIME PLASMA A PLASMA B PLASMA C PLASMA D eiinutes cent cent 89 It is generally considered that the liver is the major site for the destruction of local anesthetics, including procaine (12). These conclusions were derived from eximents in which the drugs lost their toxicity after fusion through animal livers. Also, animals with damaged livers were found to be more susceptible to the toxic actions of local anesthetics (12). On this basis, it has been suggested that their extensive use should haps be avoided in patients with severely diseased livers. The results reported here suggest that the liver is not an important site for the transformation of procaine, at least in the human. The intravenous infusion of procaine at a rate of 20 mgm. minute resulted in plasma levels of about mgm. liter as noted above. The rate of transformation of procaine by the body may be assumed to equal the infusion rate since the urinary excretion of procaine is negligible. Assuming that the hepatic plasma flow is a liter minute and that the plasma is completely cleared of procaine at each passage of blood through the liver, the liver could hydrolyze only mgm. of the drug each minute. Since 20 mgm. of procaine are being hydrolyzed minute in the body, the compound must be transformed mainly by extra-hepatic mechanisms. The rapid rate of hydrolysis of the procaine in plasma in vitro shown above, suggests the plasma to be the main site of procaine er cent cent

8 366 B. B. BRODIE, P. A. LIEF AND R. POET break-down. The major factor in determining the safety of a particular local anesthetic drug may well be the speed at which it is hydrolyzed by plasma. Preliminary work indicates that local anesthetics including procaine are more stable in dog plasma than in human. It is possible that in some species, the liver may play a more important role in the hydrolysis of these esters. DISCUSSION. Diethylaminoethanol possesses a trialkylamino group. This group is also present in procaine and many other compounds which possess in common some or all of the following pharmacological proties: local anesthesia, quinidine-like action on the heart, spasmolytic action on smooth muscle, analgesia, and anti-allergic action. Examples of other compounds that also have most of these proties are demerol, benadryl and quinidine. The extreme rapidity with which procaine disappears from the plasma and the relative sistence and high concentration of diethylaminoethanol, suggests that the latter might account for some of the pharmacological proties ascribed to the parent drug. It therefore seems desirable to compare its proties with those of procaine. These studies are now in progress. SUMMARY 1. Methods are described for the estimation of procaine and its transformation products in biological fluids and tissues in vivo. 2. The first step in the metabolism of procaine after its intravenous administration to man was shown to be hydrolysis to diethylaminoethanol and p-aminobenzoic acid. The hydrolysis takes place with unusual rapidity. Di.- ethylaminoethanol is further metabolized in large part, but the nature of its transformation is not known. 3. The hydrolysis of procaine occurs mainly in the plasma. REFERENCES 1. GORDON, R. A.: J. Canad. Med. Assn., 49: 478, MCLACHLIN, J. A.: J. Canad. Med. Assn., 52:385, GRAUBARD, D. J., ROBERTAZZI, R. W., AND PETERSON, M. C.: N. Y. State J. Med., 47: 2187, BURSTEIN, C. L.: Anesthesiology, 7: 113, KIscH, B., KO5TER, H., AND STRAUSS, E.: Exp. Med. & Surg., 1: 51, Kscu, B., AND STRAUSS, E.: Exp. Med. & Surg., 1: 66, GRAUBARD, D. J., ROBERTAZZI, W., AND PETERSON, M. C.: Anesthesiology, 8: 236, BRODIE, B. B., UDENFRIEND, S., AND TAGOART, J. V.: J. Biol. Chem., 168: 327, BRODIE, B. B., AND UDENFRIEND, S.: J. Biol. Chem., 158: 705, BRODIE, B. B., UDENFRIEND, S., AND BAER, J. E.: J. Biol. Chem., 168: 299, BRATTON, A. C., AND MARSHALL, E. K.: J. Biol. Chem., 128: 537, GOODMAN, L., GILMAN, A.: The Pharmacological Basis of Therapeutics, Macmillan Co., New York, 1941, pp