THE REACTION OF ACETYLCHOLINE AND OTHER CARBOX- YLIC ACID DERIVATIVE8 WITH HYDROXYLAMINE,

AND ITS ANALYTICAL APPLICATION*

BY SHLOMO HESTRINt

(From the Department of Neurology, College of Physicians and Surgeons, Columbia Unitxrsity, New York)

(Received for publication, February 24, 1949)

In spite of its physiological importance acetylcholine has been deter- minable chemically only after isolation by time-consuming procedures. Of necessity workers in this field have resorted to pharmacological bio- assays whose specificity seems open to doubt. The finding that choline is readily acetylated by the action of a coenzyme-linked enzyme system in which adenosine triphosphate serves as an energy source (1) lends added interest to the development of adequate analytical procedures for acetylcholine.

This communication concerns certain properties of the interaction of scetylcholine with hydroxylamine, and the means of using this reaction, RCOOR’ + HzNOH ---f RCONHOH + R’OH, for the quantitative de- termination of acetylcholine. Feigl, Anger, and Frehden (2) have de- scribed the use of hydroxylamine as a spot test reagent (cf. also (3)). Lipmann and Tuttle (4) have based a specific quantitative method for the acyl phosphates on their ability to react with hydroxylamine in water at pH 6. Hill (5, 6) has used the reaction of est,ers with hydroxylamine at alkaline pH in anhydrous solvents to determine long chain fatty acid esters (cf. also (7)). The present method, which is designed for use with short chain 0-acyl derivatives, is based on the finding that hydroxylamine at an alkaline pH in water rapidly converts acetylcholine stoichiomet- rically to hydroxamic acid throughout a wide range of ester concentration. The specificity of the reaction as between esters and products of t.heir hydrolysis and its rapidity and technical simplicity have afforded an analytical method which is adaptable to widely different conditions.

Hydroxylamine has been employed a,s a trapping reagent for acyl an- hydrides in mixed enzyme systems by Lipmann and Tuttle (8) and others (9-11). Chantrenne (12) has shown that hydroxylamine at pH 7.1 also traps carboxylic acid esters. Experiments reported below evaluate the

*This work has been carried out under grants from the United $States Public Health Service and the Office of Naval Research.

t Present address, The Hebrew University, Jerusalem. 249

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250 REACTION OF ACETYLCHOLINE

effect of reactant concentration and pH on the rate of this reaction in the case of acetylcholine.

Method

Reagents 1. Hydroxylamine. Hydroxylamine hydrochloride, 2 M. The solution

should be stored in the cold. 2. Alkali. Sodium hydroxide, 3.5 N.

3. Acid. Concentrated hydrochloric acid, sp. gr. 1.18, diluted with 2 parts by volume of water.

4. Iron. Ferric chloride, 0.37 M, in hydrochloric acid, 0.1 N. Reagent iron chloride, ferric (FeCl,.GHyO), crystals, of Merck and company was used.

5. Standard solution. Acetylcholine chloride, 0.004 M, in sodium ace- tate solution, 0.001 N, of pH 4.5. This standard may be kept in the refrigerator for a fortnight without measurable loss.

Procedure for Determination of Acetglcholine and Related Esters-Alkaline hydroxylamine reagent is prepared freshly before use by mixing equal volumes of Reagents 1 and 2. The mixture keeps for about 3 hours at room temperature. 2 ml. of alkaline hydroxylamine reagent are added in a test-tube to 1.0 ml. of the solution to be analyzed. After at least 1 minute, or longer if desired, the pH is brought to 1.2 f 0.2 with 1.0 ml. of acid, and 1.0 ml. of the iron solution is added. The density of the purple-brown color is promptly determined at 540 rnp. Formation of gas bubbles in the calorimeter cell is avoided if the mixture has been swirled adequately after the addition of each component. Solutions too dark to read are brought to suitable density by diluting with ferric chloride, 0.074 M, in 0.1 N hydrochloric acid. If the color reading is low, the analysis may be carried out on a larger ester aliquot (up to 2.5 ml.) with reagents of a suitably higher concentration. Correction for non-specific color is made by repeating the procedure as described, except that the order of addition of hydroxylamine, alkali, and acid is reversed. With this order of addition of the various components, esters do not form any hydroxamic acid.

Protein in the sample analyzed is generally precipitated when the acid and iron additions are made and may be removed either by filtration or brief centrifugation. Formation of a flocculent precipitate may be hzs- tened by the addition of trichloroacetic acid.

The extinction, after being corrected for non-specific absorption, is converted to concentration with the help of a proportionality factor which is found by applying the same procedure to a standard solution. When buffer is present in the unknown, the strength of the added acid component

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8. HESTIZIN 251

should be adjusted to compensate for the effect of the buffer on the pH If the ester sample contains iron-binding buffer salts, an increase in the iron concentration may be used to insure the maximum development of color. If necessary, a correction is applied for the effect of buffer salts on the optical density. In a case in which the color density of the blank is undesirably high, the efficacy of lower iron concentration or lower final pI-1 t,han indicated as a means of reducing the relative color density of the blank should be determined.

The upper limit of measurement exceeds 5 PM and the lower limit is about 0.04 PM of ester per ml. of final solution.

Results

Except in so far as specified below, the conditions described under the procedure were used.

The reaction with hydroxylamine was carried out in 2 minutes at room temperature in solutions which contained (micromole per ml.) 660 of hydroxylamine, 6G0 of sodium chloride, 500 of sodium hydroxide, and 2 or less of ester. An aliquot of 3.0 ml. was brought to pH 1.0 with 1.0 ml. of hydrochloric acid and to a final iron concentration of 37 PM per ml. with 1.0 ml. of ferric chloride, 0.185 M, in 0.1 N hydrochloric acid.’ The density of color was measured immediately to minimize error from fading. The Klett-Summerson photoelectric calorimeter was used with green filter No. 54. Extinction values were corrected for the blank absorption.

Observations on Ferric-Acethydroxamic Acid Complex-The color of the ferric complex of acethydroxamic acid is analyzed by Fig. 1. In the range X = 520 to 540 rnp, little or no absorption is shown by ferric chloride, while absorption by the ferric-acethydroxamic acid complex is nearly maximum. As is shown by Fig. 2, the color density is essentially inde- pendent of pH between 1.0 and 1.4 and decreases with pH between 1.0 and 0.6. The color o!ten faded somewhat on standing. The stability appears to depend on pH. In one experiment, for instance, fading ac- counted for about 5 per cent of the initial color after 10 minutes at pH 1.0, and for about 12 per cent at pH 1.4. The red color formed by acetic and propionic acids with ferric chloride was found to be relatively weak at any pH and was suppressed completely at pH 1.2. As much as 7 rnM of sodium acetate or propionate showed no color and failed to alter the color reaction given by 2 PM of acetylcholine chloride at this pH. Choline chloride in amounts up to 3.5 mM failed to affect significantly the reaction obtained with acetylcholine in the absence or presence of propionate or acetate.

1 It was eventually found that 74 PM of iron per ml. would have been preferable for the analysis.

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252 REACTION OF ACETYLCHOLINE

The variation of color density with iron concentration is illustrated by Curve 1 of Fig. 3. Since considerable molar excess of iron is needed for full color development, and since the approach to maximum color is asymp- totic, the dissociation tendency of the ferric-acethydroxamic acid complex must be fairly high at the pH used (cf. (13)). With an iron concentration of 74 PM per ml. as used in the procedure, the color may be estimated to be about 94 per cent of maximum.

Absorption by ferric chloride blank solutions containing iron concen- trations of 37 or 74 PM per ml. was at the most equivalent to 0.02 to 0.03

700 660 620 560 540 500 460 420

FIG. 1. Absorption of visible light by ferric-acethydroxamic acid complex. Ace thy- droxamic acid formed from 1.5 PM of acetylcholine in 5.0 ml. of final solution. Extinction determined with the Beckman spectrophotometer. X, reagent blank; 0, total extinction; broken line, absorption by ferric-acethydroxamic acid complex (calculated by difference).

PM of ester per ml. of final mixture when measured with a Beckman spec- trophotometer and to 0.03 to 0.04 PM when measured with the Klett instrument. With 150 PM of iron, absorption by t,he blank solution was nearly twice as high.

The inhibitory effect of certain iron-binding anions (phosphate, sulfate, fluoride, oxalate) on the color reaction has been noted by Lipmann and Tuttle (4). Further information concerning the phosphate effect is pro- vided in Fig. 3. The convergence of the curves corroborates the view that phosphate exerts an inhibitory effect on the reaction, because it competes with hydroxamic acid for iron. The data show that the effect of phos- phate is suppressed completely if a suitable excess of iron is allowed. Under the conditions described in the procedure, the interfering effect of a total of 50 PM of phosphate was not more than 3 per cent in a tested

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S. HESTRIN 253

range of 0.2 to 2.0 PM of ester in 5 ml. of final mixture. In the same con- ditions, 50 to 200 PM of sulfate or 100 PM of borate produced little or no interference.

Formation of Acethydroxamic Acti in Strongly Alkaline Solution-The velocity of reaction in the standard reagent was very great and could not be measured. The maximum yield of hydroxamic acid was found after 1

0 - 0.6 ob I.0 IIf 1.4 30 60 so IL0 150

PH IRON, uM/ml. FIG. 2 FICL 3

FIG. 2. Influence of acidity on color density. The terminal pH was varied by adjusting the hydrochloric acid normality and was measured with a glass electrode. Color was developed in a solution of 5.0 ml. containing the hydroxamic acid formed from 3 PM of acetyIchoIine.

FIG. 3. Phosphate inhibition of color reaction at varying iron and phosphate concentration. Solutions contain 0.7 microequivalent of acetylcholine per ml. of final mixture. Curves 1 to 4 correspond to solutions with 0, 10, 30, and 50 PM of orthophosphate per ml.

minute at room temperature and remained constant thereafter for at least 5 hours. When applied to an enzyme reaction syst,em, t,he alkaline reagent &us brings about, both the rapid inactivation of the proteins and instantaneous transformation of the ester in the sample to a product which can conveniently be stored before it is determined. This finding is of interest in regard to the mechanism of the reaction. Since the hydrox- amic acid level stays constant, the reaction in alkaline medium must be considered essentially irreversible.

Representative results relating acetylcholine concentration to color in-

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2.54 REACTION OF ACETYLCHOLINE:

tensity are plotted in Fig. 4. The yield of hydroxamic acid is shown to be a direct function of acetylcholine concentration. Similar results were found also with other esters tested; e.g., propionylcholine, triacetin, and ethyl acetate.

It is noteworthy that the concentration density curves all pass through zero and that the yield of hydroxamic acid at termination of reaction is independent of the pH of reaction within a wide range (pH 9.2 to 13.3). Hydroxylamine buffered with sodium carbonate to pH 9.2 has been used by us in fact to determine acetylcholine in the presence of eserine. The

ACETYLCHOLINE, yM per ml. ester sol. FIG. 4. Density of color as a function of acetylcholine concentration. Curves

1 and 2 were obtained with a Klett photoelectric calorimeter, Curve 1 with ordinary reaction conditions; Curve 2 with hydroxylamine at pH 9.2 and allowing 60 minutes for completion of the reaction of acethydroxamic acid formation. Curve 3 was obtained with a Beckman spectrophotometer at X = 540 rnp on a solution layer of 1.0 cm. The iron concentration was 37 ,UM per ml. in every case.

fraction of acetylcholine which undergoes hydrolysis in the presence of the hydroxylamine reagent could only be either negligibly small, or, alterna- tively, constant for a wide range of pH and ester concentration. The latter alternative appeared unlikely. When the hydroxylamine concen- tration was raised from 0.66 to 1.0 M, the end yield of hydroxamic acid remained unchanged. This supports the conclusion that the transforma- tion of acetylcholine in alkaline medium proceeds with much greater rapidity in the direction of hydroxamic acid formation than in the direc- tion of hydrolysis, since hydrolysis, if significant, would have become evident in the form of diminished yield of hydroxamic acid at the lower hydroxylamine concentration.

Factors Affecfing Formation of Hydroxamic Acid i?a Physiological pH Range

pH-The effect of the pH on the rate of reaction of acetylcholine with hydroxylamine is illustrated by the experiments summarized in Table I.

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8. HESTIUN 255

The data show that the reaction is slow but eventually complete even at pH 6.4 and that the rate rises with increasing rapidity as the pH is in- creased. The controlled use of hydroxylamine as a trapping agent is dependent on recognition of this Ale of the pH. To trap esters as well

TABLB I Effect of pH on Reaction of Acetylcholine with Hydroxylamine

Concentration of hydroxylamine and acetylcholine, 800 and 2.1 pM per ml. pH adjusted with sodium carbonate. Temperature 37.5”. Aliquots of 2.5 ml. were brought to pH 1 at the times specified and analyzed for hydroxamic acid in a final volume of 5 ml. The initial reaction rates were found by extrapolation.

! Hydroxamic acid in microequivalents of acetylcholine after I

PH - 2 min.

____-

7 5 min.

T

0 min. 11 .-

-

2.90 4.95

5 min. 30 min. --

0.76 1.57 2.15 4.20 5.15 5.20 5.15 5.15

0 min.

- I

3.15 5.15

-

___ Initial rate of 120 300

I 1330 reaction

min. .min. min. .

1.50 4.95 5.15

-

3.08

jIM x 100 per min.

4.60 1.2 5.2

14.3 29.0

114.5

TABLE II Formation of Hydroxamic Acid at Various Concentrations of Net&al H?)droxylamine

Hydroxamic acid formed from 5.6~~ of acetylcholine in 2.5 ml. of reaction mixture containing the indicated concentrations of hydroxylamine at pH 6.9. Temperature 37”.

Concentration Hydroxamic acid in microequivalents of acetylcholine after of NHZOH

(0) 35 min. 60 min. 120 min. 800 min.

#Y per ml.

3200 4.57 5.57 5.37 1600 3.47 4.67 800 2.09 3.46 5.76 400 0.98 1.93 4.58 200 0.36 0.61 2.31

Time of one-quarter

reaction (b)

min.

11 20 40 85

325

(bf X (01 -- 10’

35 32 32 34 65

as other reactive acyl derivatives, the pH of the mixture should be adjusted to the maximum value compatible with maintenance of the enzymatic activity; to trap acyl anhydrides selectively, the pH should be lowered.

Concentration of Hydroxylamine-The effect of the concentration of hydroxylamine is shown in Table II. In this experiment the concentra- tion of hydroxylamine was varied from 0.2 to 3.2 M, while the molar ratio of hydroxylamine to acetylcholine exceeded 90 throughout. The time

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256 REACTION OF ACETYLCHOLINE

required for 25 per cent conversion of acetylcholine to hydroxamic acid is found to be an inverse linear function of t>he hydroxylamine concentration between 0.4 and 3.2 Y. With high hydroxylamine concentrations, the yield of hydroxamie acid rapidly approached a maximum which did not differ from that found with the same quantity of acetylcholine in alkaline reagent. With the lowest ooncen6ration of hydroxylamine tested, on the ot,her hand, the reaction became very slow at a stage in which a major part of the acetylcholine had not yet reacted. This result is consistent with the conclusion of Chantrenne (12) that t.he selectivity of hydroxyl- amine as a trapping reagent for acyl anhydrides is improved if the concen- tration of hydroxylamine is lowered.

TABLE III Formation of Hydroxamic Acid at pH 7.1 from Various Concentrations of Acetylcholine

A solution containing hydroxylamine 0.61 M, choline chloride 1.35 M, and sodium acetate 1.35 M was incubated at pH 7.1 and 37.5” with acetylcholine in the specified concentrations. At the times indicated, 1.0 ml. ‘aliquots were brought to pH 1.0 and analyzed. Slight formation of hydroxamic acid occurred by reaction of hydrox- ylamine with free acetate. The values reported have been corrected for this side reaction.

I Concentration of )

Hydroxamic acid in microequivalents of acetylcholine after

acetylcholine I ----

____--- 15 min. I 60 min. / 120 min.

p&f per ml. j I

8.8 1.10 4.14 6.12 / 4.4 0.62 1:99 2.70 2.2 I 0.31 I 0.98 1.39

Concentration of Acetylcholir?e-The course of the reaction wit.h varying initial concentrations of acetylcholine is illustrated by Table III. The data show that the velocity of reaction in excess of hydroxylamine is a direct function of acetylcholine concentration.

0-Acyl Derivatives-Findings with a randomly assembled collection of O-acyl substances, including thirty-five esters and a few lactones and anhydrides, are shown in Table IV. It is evident that a number of the esters fail to respond to the test in its present form.

The data suggest that the molar color yield of esters remains independent within certain limits from the chain length of the acid radical. Thus,

2 I am much obliged to Dr. D. Shemin and Dr. H. Waelsch for amino acid and peptide esters, to Dr. D. Rittenberg for cholesterol esters and diacetylglycine diketa- piperazine, and to Dr. J. Aeschlimann for miscellaneous groups of esters.

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S. HESTRIN 257

TABLE IV Chromoyenic Activity of 0-Carboxylic Acid Derivatives

Tests were carried out with approximately 5 MM of the substance, allowing a reac- tion time of 4 minutes. Water-insoluble substances and anhydrides tested were added to the reagent in 0.1 ml. of methanol. The latter does not affect the color reaction at this concentration.

Chromogenic activity per 0-acyl equivalent relative to

acetylcholine - 100 -.-.__-___._

Esters of simple monocarboxylic acids

Ethyl formate. ................... n-Propyl formate. ................ Ethyl acetate ..................... Triacetin ......................... Glucose pentacetate. .... :. ....... @-Dimethylaminoethyl acetate. ... Acetyl-&methylcholine (mecholyl) Cholesteryl acetate. ....... .... Ethyl propionate. ................ Propionylcholine. ................ Butyrylcholine. ................... Tributyrin ........................ Methyl butyrate .................. Cholesteryl butyrate. ............. Ethyl n-caprylate. ................ Peanut oil. ....................... Sorbitan oleate (Tween 80). ....... Tripalmitin. ......................

......

......

......

......

. . . . . .

...... ...... ...... ...... . . . . . . ...... ...... ...... ......

.

..,...

. . . . . .

......

...... ...... ...... . . . . . . ...... ...... ...... ......

......

......

......

...... . . .

......

......

......

I . .../ 36 I

‘.“I 41 ./ 97

“.‘/ 98 . . , 110

. 116 . 107 . . 0*

. 95 . . . 101

“‘./ a7 95

. 87 . . 0’

. . . 78 . . 0

Esters of complex or cyclic monocarboxylic acids

Benzylbeenzoate ........................................ Methyl p-hydroxybenzoate (nipagin).

..~ .....................

p-Aminobenzoic acid ester of 2,2-dimethyl-3-diethylamino- i

propanol (larocaine) .................................... ~~r-Pyrrolidonecarboxylyl-p-aminobenzoic acid ester of eth-

an01 .................................................... K-pyrrolidonecarboxylylglycine ester of ethanol. .......... dl-Tropic acid ester of 3-diethylamino-2,2-dimethylpropanol

(syIltropan) /

........................................... ..j y-Ethyl ester of glut,amic acid ............................ .I Carbethoxylanylglycine. .................................. Dimethyl carbamic acid ester of 3-hydroxyphenyltrimethyl

i

ammonium bromide (prostigmine). /

..................... .I Carbaminoyl choline. ..................................... Ethyl ester of 4-phenyl-1-methyl-4-carboxylpiperidine (dem-

erol) ....................................................

97 28

5

106# 75

58 7 0

0 0

0

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258 REACTION OF ACETYLCHOLINE

TABLE IV-Concluded

Chromogenic activity per 0-acyl equivalent relative to

acetylcholine = 100

Polyesters of polycarboxylic acids

Diethyloxalate ........................................ .../ 23 ‘I malonate ...................................... ...’ 58 “ succinate ......................................... “ oxalacetate ....................................... 1 & .

Carbethoxyalanylglycine ethyl ester ....................... 40 Triethyl ester of r-glutamylglutamic acid ................. 42

Lactones

0-Coumaric acid lactone (coumarin) . . . . . . . . d-Pantoyllactone.........................................

Carboxylic acid anhydrides

851§ 106

Benzoic acid anhydride. . . . . . . . . . . . . . . . . . . . . . . . . Succinic “ “ . . , . . . . , . . . . . . . . . . . . . . . . Phthalic ‘I “ . . . . . . . , . , . . . . . . . . . . . . . , . . . . . . . . ,

loot 97 658

-. * Failed to dissolve. t Mixture warmed to dissolve. $ Greenish. 8 Color fades rather rapidly.

esters of acetic, propionic, and butyric acids, provided that they could be dissolved in the reagent, all yielded an equivalent amount of color (cf. (4)). Two formic acid esters and a caprylic acid ester, however, gave lower readings. On the other hand, the fat specimens, i.e. esters of long chain fatty acids, failed to yield the color reaction under the same condi- tions. This finding is noteworthy in connection with the proposed applica- tion of the reagent, in the presence of blood or tissue preparations. Long chain fatty acid esters are able to form the characteristic color with ferric iron if the reaction with the hydroxylamine is carried out with warming in an anhydrous organic solvent, (5,7).

As is to be expected, substituents of the acid radical may influence the color yield. The tested carbamic acid esters, for example, failed to yield the color. With suitably placed polar groups in the acid radical, more- over, pronounced depressant effects on the color yield were observed; e.g., -NH2 or -OH in the para position in the benzoyl esters. The relatively low chromogenic activity per ester linkage of total esters of the polycarboxylic acids provides a further example of the effect of a rival polar group.

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S. HESTRIN 259

Esters of short chain fatty acids containing widely different alcohol radicals reacted with the hydroxylamine reagent apparently with equal facility. Only the two cholesterol esters, neither of which could be dis- solved in the reaction mixture, failed to react. Lactones, i.e. internal esters, reacted in the manner of ordinary esters. Carboxylic acid an- hydrides, a group known to react readily with hydroxylamine at acid pH, reacted readily also with the alkaline reagent.

N-Acyl Derivatives arxd Other Compounds-Bergmann, du Vigneaud, and Zervas (14) have directed attention to the marked activity of diacetyl- glycine diketopiperazine as an acctyl donor for amino acids. In the conditions of the ester assay, this substance reacted almost quantita- tively with hydroxylamine (chromogenic activity per acetyl = 90). Com- parable reactivity with hydroxylamine on the part of an N-acyl deriva- tive appears also to be exhibited as the /3-lactam group of penicillin and has been used in fact for the determination of penicillin (15).

Other N-acyl derivatives tested, including amides (acetamide and as- paragine), acylated amino acids (acetylglutamic, acetylphenylalanine, hip- puric, and pantothenic), peptides (glutnthionone and cr-glutamylglutamic acid), alanine diketopiperazine, acetylglucosamine, and acetylsulfathiazole failed to yield a significant amount of color (chromogenic activity per acyl group <3) in the conditions which permitted the quantitative reac- tion of 0-acyl derivatives (reaction time 1 minute at room temperature).

It should be noted that hydroxylamine reacts fairly readily with certain ketonic substances. The resulting oximes may yield a color reaction (9). Comparatively large amounts of sodium formate, acetate, propionate, and barbiturate failed to form color and did not interfere with acetyl- choline estimation. Adenyl pyrophosphate, tetraethyl pyrophosphate, as- corbic acid, betaine, and gelatin in a concentration of 0.2 mg. per ml. of color solution did not form any color. Cysteine forms a blue ferric com- plex, which fades rapidly to green. An interference by cysteine in the measurement of acetylcholine was only encountered at cysteine concen- trations greater than 20 PM per ml. of final solution.

DISCUSSION

The method which has been described here was primarily designed for the estimation of acetylcholine synthesis by choline acetylase. It is par- ticularly suited for determination of ester synthesis in the presence of an excess of products of ester hydrolysis. Actually the method has proved convenient not only for choline acetylase, but equally for cholinesterase, assay.

Calorimetric measurement of esterase activity in terms of disappearance of acetylcholine supplements the known manometric and titrimetric assay

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260 REACTION OF ACETYLCHOLINE

techniques, which are based on measurement of the acid formed. Iden- tical results were found when acetylcholine hydrolysis in bicarbonate buffer was determined calorimetrically and manometrically. Since the reaction rate of hydrolysis is measured in the calorimetric method from the dif- ference of successive decreasing readings, the calorimetric procedure is relatively inaccurate, while the fraction of substrate hydrolyzed is small, but provides an accurate and convenient means for following the total course of the hydrolysis reaction. The compensating merit of the colori- metric method is its adaptability to widely different experimental condi- tions. The reaction kinetics of esterase can be studied by this method with a uniform procedure over a wide range of pH, substrate, and enzyme concentration, in almost any buffer and not merely in bicarbonate, and in systems in which acid or alkali is formed in the course of the experiment by a concomitant side reaction. The method has been employed to determine the equilibrium constant of acetylcholine hydrolysis. Experi- ments which show these applications are to be described in forthcoming communications.

Since a variety of carboxylic acid derivatives react with hydroxylamine, the range of the processes for which the met,hod may offer a rapid and simple technique of determination is wide. Int,eresting possibilities are apparent in connection with the use of hydroxylamine to determine blood and tissue concentrations of drugs which have the structure of esters, e.g. coumarin. Acetylcholine added to human blood serum or to hemol- ysate of red blood cells was recovered analytically without loss. Hy- drolysis of the added ester in these media could be followed readily. The serum and hemolysate blank colors were negligible. The proteins were precipitated by the reagent mixture at acid pH and removed by filtration without retention of hydroxamic acid in the precipit.ate in an absorbed form.

Hydroxamic acid formation takes place in absence of adenosine triphos- phat,e when hydroxylamine is added in adequate concentration together with fatty acid to a liver homogenate (8, 12). Lipmann (16) has sug- gested that this acylation is mediated by lipases (esterases). The observa- tion that esters readily acylate hydroxylamine in a physiological range of pH made it possible to assume that esters, synthesized by reversible es- terase activity, could have played an intermediary role in the hydroxamic acid formation (12). The conclusion that esterases mediate the N-acyla- tion directly has, however, been supported by experiments, as yet unpub- lished, which have shown that hydroxamic acid is formed when purified acetylcholine esterase is incubated with hydroxylamine and sodium acetate.

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S. HESTRIN 261

SUMMARY

1. A rapid micromethod for short chain carboxylic acid esters, lactones, and anhydrides is based on their ability to react with hydroxylamine quantitatively in an aqueous alkaline solution.

2. Acetylcholine may be determined by t,he method in the presence of large excess of acetate and choline.

3. Conditions which determine the scope of action of hydroxylamine as a trapping reagent have been investigated.

It is a privilege to thank Professor D. Nachmansohn for his kind in- t’erest and advice in the conduct of the investigation. The technical as- sistance of Mrs. Emily Feld-Hednl is acknowledged gratefully.

BIBLIOGRAPHY

1. Nachmansohn, D., and Weiss, M. S., J. Biol. Chem., 173, 677 (1.948). ’ 2. Feigl, F., Anger, V., and Frehden, O., dlikrochemie, 16,9 (1934).

3. Davidson, D.,.Z. Chem. Education, 17,81 (1940). 4. Lipmann, F., and Tuttle, L. C., J. Biol. Chem., 169.21 (1945). 5. Hill, U.,Znd. and Eng. Chem., Anal. Ed., 18,317 (1946). 6. Hill, U., Anal. Chem., 19,932 (1947). 7. Bauer, F. C., Jr., and Hirsch, E. F., Arch. Biochem., 20, 242 (1949). 8. Lipmann, F., and Tuttle, L. C., J. Biol. Chem., 161,415 (1945). 9. Speck, J. F., J. Biol. Chem., 168,403 (1947).

10. Elliot, W., Nature, 161,128 (1948). 11. Elliot, W., and Gale, E., Nature, 161,129 (1948). 12. Chantrenne, H., Compt. rend. trav. Lab. Curlsberg,%, 231 (1948). 13. Hantzsch, A., and Desch, C., Ann. Chem., 323,l (1902). 14. Bergmann, M., du Vigneaud, V., and Zervas, L., Ber. them. Ges., 62,1909 (1929). 15. Clarke, H. T., Johnson, J. R., and Robinson, R., The chemistry of penicillin,

Princeton, 428,1029 (1949). 16. Lipmann, F., Advances in Enzymol., 6,257 (1946).

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Shlomo HestrinANALYTICAL APPLICATIONHYDROXYLAMINE, AND ITS

DERIVATIVES WITHAND OTHER CARBOXYLIC ACID

THE REACTION OF ACETYLCHOLINE

1949, 180:249-261.J. Biol. Chem. 

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