The formation of α-acetolactic acid and acetylmethylcarbinol by Ascaris lumbricoides

EXPERIMENTAL

PARASITOLOQY

7,

477499

(1958)

The Formation of wAcetolactic Acid and Acetylmethylcarbinol by Ascaris lumbricoides’ Howard

J. Saz, Arthur

Department

Vidrine,

Jr.,2 and Jeanette

A. Hubbard

of Pharmacology, School of Medicine, Louisiana University,

State

New Orleans, Louisiana

(Submitted for Publication, 3 January 1958) The formation of acetylmethylcarbinol (AMC, acetoin) has been demonstrated in bacteria, yeast, animal tissues, and plants, as well as in normal human urine (for references see Berl and Buedmg, 1951). Berl and Bueding (1951) have shown that the Maria1 nematode, Ldmwsoidescar&ii also produces AMC. Juni (1952) elucidated the bacterial mechanism of AMC formation by the isolation of two enzyme systems from Aerobacter aerogenes.One enzyme catalyzed the synthesis of cu-acetolactic acid from pyruvic acid (reaction 1) ; the other enzyme catalyzed the decarboxylation of a-acetolactic acid to form AMC (reaction 2). (1) (2)

2 CHaCOCOOH

-+ CHaCOH(COCHa)COOH

CHpCOH(COCHa)COOH

-+ CHsCHOHCOCHa

+ CO* + CO2

This mechanism has been demonstrated only in bacteria (Juni, 1952; Dolin and Gunsalus, 1951). Subsequently, Juni and Heym (1956) have presented data in support of a second mechanism of AMC formation. These authors postulated that pyruvic oxidase catalyzes the decarboxylation of pyruvic acid to form carbon dioxide and an acetaldehydediphosphothiamine-magnesium complex. This “active” acetaldehyde may then condense with either pyruvic acid to form a-acetolactic acid or with free acetaldehyde to form AMC directly. In the course of studying the metabolism of Ascaris lumbricoides it 1 This investigation was supported in part by the National Science Foundation, grant No. G 2491. 2 Supported by a medical student summer research award from Smith, Kline and French, Inc. 477

478

SAZ, VIDRINE

AND HUBBARD

was found that this nematode formed AMC as a normal metabolic product. The mechanism by which this compound is formed was further investigated. MATERIALS

AND METHODS

Ascaris lumbricoides var. suis was obtained from a local slaughterhouse and transported to the laboratory at room temperature in a physiological salt solution containing antibiotics (Epps et aE., 1950). Muscle strips were collected as described by Laser (1944) and either used directly or frozen and stored at -20°C. No loss of AMC forming ability was detected after storage of the muscle for periods of up to several months, Crude enzyme preparations which were capable of forming AMC were prepared from muscle strips of Ascaris Zumbricoides.The muscle was cooled and minced in a beaker surrounded by chopped ice. The mince was homogenized with three volumes of 0.04 M tris (hydroxymethyl) aminomethane buffer, pH = 8.9 in all glass Potter-Elvehjem homogenizer. The mixture was centrifuged at 25,000 X g for 30 minutes. The supernate was collected and employed without further purification. All operations were performed between 04°C. Precipitation of proteins was accomplished according to Somogyi (1930). AMC was determined by the micromethod of Westerfeld (1945). However, a slight modification of this method was necessary when manganese was present in the filtrate, since this metal interfered with the color development (Berl and Bueding, 1951). Therefore, the manganese was precipitated by the addition of alkali lo-15 minutes prior to the addition of the zinc sulfate solution. The dimer of AMC was obtained commercially, washed with ether and dried in vacua at room temperature. a-Acetolactic acid was synthesized according to the method of Krampitz (1948)3. a-Acetolactic acid was estimated by decarboxylating the compound in the presence of mineral acid and determining the increase in AMC formation. After deproteinization, the filtrate was divided into two aliquots. One aliquot was used directly for the determination of AMC. Sulfuric acid was added to the second aliquot until it was approximately 1.7 N with respect to this acid. The solution was then placed in a boiling water bath for 5 minutes, cooled in an ice bath and neutralsThe authors wish to thank Dr. E. Juni, Emory University, for a generous gift of the ethylacetoxy ester of cu-acetolacticacid.

ACETYLMETHYLCARBINOL

FORMATION

BY

479

ASCARIS

ized with sodium hydroxide. Then the AMC content of this aliquot was determined. The increase in AMC was attributed to the decarboxylation of ar-acetolactic acid with the concomitant formation of AMC. 1,2-Naphthalenediamine was synthesized according to Bamberger and Schieffelin (1889) and purified by sublimation according to Berl and Bueding (1951). The dihydrochloride was prepared by dissolving the base in excess HCl. All other preparations employed in this investigation were obtained commercially. Acetaldehyde and diacetyl were distilled immediately before use. Unless otherwise stated, all enzymatic reactions were incubated in the conventional Warburg respirometer. RESULTS

Formation

of AMC

by Intact Ascaris

lumbricoides

Two adult female ascarids were placed in each of five flasks containing 50 ml of a physiological salt solution (Epps et al., 1950), 3.75 mg streptomycin, 5 mg penicillin, 25 mg gantrisin (sulfisoxazole), 0.5 mg Mycostatin (nystatin), and the substrates indicated in Table I. After incubating the vessels at 37” for 19 hours, an aliquot was removed from each flask and centrifuged at 1400 X g for 20 minutes to remove any eggs which may have been present. The AMC content of each aliquot was then determined. From the results tabulated in Table I, it can be seen that AMC was formed in each flask. No increase in AMC production over endogenous was observed upon the addition of glucose as substrate. This may be TABLE Production

of AMC

Molar Glucose

concentration

by Zntact

1 Ascaris

lumbricoides

of substrate

sodium pyruvste

Acetaldehyde

3 x 10-2 3 x lo-2 3 x 10-f *fig per gram of Ascaria lumbricoidss For further detaila sea tat.

(wet weight)

1.5 x 10-2 1.5 x 10-2

AMC

formed La’

10.6 8.1 13.4 64.3 121.3

480

SAZ,

VIDRINE

AND

HUBBARD

explained on the basis of the high glycogen content of these helminths. The glycogen would serve as a source of glucose inside the ascarid which may saturate one or more of the enzymes involved in AMC formation. Therefore, the addition of exogenous glucose would have little effect. As was found to be the case in Litomosoides car&ii (Berl and Bueding, 1951), AMC formation was approximately five times greater from acetaldehyde than from pyruvate. When both acetaldehyde and pyruvate were present in the medium there was approximately ten times more AMC formed than from pyruvate alone. To be certain that the antibiotics and salt solutions employed in this experiment were not chromogenic in the calorimetric AMC determination, one additional control flask was employed. This vessel contained the same incubation mixture as the other five flasks with the exceptions of substrate and helminths. It was incubated concurrently with the other vessels. No AMC was detectable under these conditions. Formation of AMC and ar-Acetolactic Acid by Homogenates of Ascaris Muscle

Crude enzyme preparations were prepared as described above. The data recorded in Table II illustrate that such homogenates catalyze the formation of both AMC and a-acetolactic acid. The relative quantities of AMC formed from pyruvate, acetaldehyde, or pyruvate plus acetaldehyde agree with the results obtained with intact helminths. In contrast to AMC, more a-acetolactic acid is formed from pyruvate than from acetaldehyde. However, in the presence of both pyruvate TABLE II AMC and cu-Acetolmtic Acid Formation by Homogenates of Ascaris lumbricoides Muscle Molar sodium pymvate

concentration

of substrate

Acetaldehyde

0.1 0.1 0.1

1.5 5 1.5 5

x x x x

lolo-’ 10-Z lo-’

AMC

formed M

16.7 84.8 63.2 134.4 680 680

Eae.h flask contained 6 molea of M&h, 0.4 ml of enzyme, of sodium a&ate btier, pH = 6.6 and sub&ate as indicated. at 3i’O for 2 houm; gm pbam N, .

a-A&ok&c

acid

formed

rg

0 19.2 0 9.6 40 48 0.3 mg of diphmphothiamim, 160~01~ Total volume, 2.0 ml; all vessels shaken

ACETYLMETHYLCARBINOL

FORMATION

BY

ASCARIS

481

and acetaldehyde, maximal quantities of ar-acetolactic acid are formed. These data are in accord with the mechanism postulated by Juni and Heym (1956) to the effect that “active” acetaldehyde can condense with either free acetaldehyde to form AMC or with pyruvate to form OLacetolactic acid. An increase in a-acetolactic acid would be expected when both pyruvate and acetaldehyde are employed as substrates since the addition of free acetaldehyde was found to accelerate the rate of pyruvate decarboxylation by pyruvic oxidase preparations (Green et al., 1942). The fact that some a-acetolactic acid was formed from the higher concentration of acetaldehyde alone is presumably due to the presence of small amounts of pyruvate or a precursor of pyruvate in the crude enzyme preparation. Figure 1 illustrates the rate of formation of AMC from pyruvate.

TIME (minute*)

FIG. 1. Rate of AMC formation from pyruvate by Ascotis muscle homogenate. Each flask contained 5 pmoles of MnClr ,0.4 ml of enzyme, 0.3 mg of diphosphothiamine, 160 pmoles of sodium acetate buffer, pH = 5.6 and 200 pmoles of sodium pyruvate. Total volume, 2.0 ml; all vessels shaken at 37’; gas phase N, . Endogenous values have been subtracted.

482

SAZ,

VIDRINE

AND

HUBBARD

ACE1A-E

SUFFER

PO4 BUFFER

01 4.6

,,,,,,,,,,,,,, 51)

6.4

6.6

62

6.6

1.0

7.4

’

pn FIQ. 2. pH optima curves for AMC formation by Ascan’s muscle homogenate in acetate and phosphate buffers. Conditions as recorded in legend to Fig. 1. Each flask contained 160 wmoles of the indicated sodium acetate or sodium phosphate buffer.

As can be seen, the quantity of AMC formed was linear with time for 2.5 hours indicating that the enzyme system was quite stable under the experimental conditions. The pH optima curves employing phosphate and acetate buffers are recorded in Fig. 2. Apparently, the nature of the buffer had a considerable effect upon AMC production since AMC formation was appreciably lower in the presence of phosphate. The pH optimum in the presence of acetate buffer was quite sharp. At a pH either above or below 5.6, the enzyme activity decreased rapidly. However with phosphate buffer, the pH optimum is not nearly so well de6ned, but the activity of the enzyme system was reduced markedly. Cofactor Requirementsof AMC Forming System Figure 3 illustrates the stimulatory effect of diphosphothiamine (cocarboxylase) on the production of AMC by muscle homogenates. Small

ACETYLMETHYLCARBINOL

0-j 0

I 0.02

FORMATION

I a04

I oila

DIPHDSPm)THIAMINE

o!ce

BY

I alo

CQNCENTRATIDN

ASCARIS

483

I 0.12 (mg./ml.)

FIQ. 3. The effect of diphosphothiamine concentration on AMC formation by Ascam’s muscle homogenates. Except for indicated diphosphothiamine concentrations, conditions were aa recorded in legend to Fig. 1. Endogenous values have not been subtracted.

quantities of AMC are formed without the addition of diphosphothiamine. It is very likely that the crude enzyme preparations employed contained some diphosphothiamine which would allow for the formation of small quantities of AMC in the absence of added cocarboxylase. Maximal stimulation was obtained by the addition of approximately 80 pg of cocarboxylase per milliliter of reaction mixture. In addition to the requirement for diphosphothiamine, the system also has an apparent requirement for manganese ions. This is illustrated in Fig. 4. The addition of Mn++ resulted in a twofold increase in the amount of AMC formed. MgH could not replace Mn++ although a very slight stimulatory effect was observed upon the addition of MgH. It is of interest that the nematode Ldomosoides car&ii also requires Mn++ rather than Mg++ for the optimal production of AMC from pyruvate (Berl and Bueding, 1951).

484

SAZ,

VIDRINE

AND

HUBBARD

60-

30

20

IO I

00 0

0.5

I.0 I.5 20 2.5 WLARITY x lo4 OF DlvALENT METAl.

3.0

FIQ. 4. The effect of manganese and magnesium ions on AMC production by Ascarie musclehomogenates. Except for indicated divalent metal concentrations, conditionswereasrecordedin legendto Fig. 1. Endogenousvalueshave not been

subtracted. E$ect of Pyruvate Concentration upon the Formation of AMC and a-Acetolactic Acid Figure 5 illustrates the relationships between pyruvate concentration and the formation of AMC and a-acetolactic acid. Maximal AMC and a-acetolactic acid formation occurs at the relatively high pyruvate concentration of 0.1 M. It is of particular interest that the curve representing cu-acetolactic acid formation so closely resemblesthe curve representing AMC formation. This suggests the possibility that cr-acetolactic acid may be a precursor of AMC as it is in the bacterial system (Juni, 1952). However, all efforts to demonstrate an cu-acetolactic acid decarboxylase in Ascuris muscle have failed. It is also possible to explain the similarities between the two curves on the basis of the common intermediate formed from pyruvate and required for the synthesis of both compounds. For example, if the decarboxylation of pyru-

FORMATION

ACETYLMETBYLCARBINOL 160

BY

ASCARIS

485

1

o: I 0

I 0.1

I 02 CONCENTRAltW

1 0.3

I 0.4

CF PYRUVATE

( Molarity)

I 0.5

FIG. 5. The effect of pyruvate concentration on AMC and a-acetolactic acid formation. Except for indicated pyruvate concentrations,conditions were as recorded in legend to Fig. 1. Endogenous values have not been subtracted.

vate or the formation of “active” acetaldehyde were rate liiting in both cases,then both curves would reflect this limiting reaction. Higher concentrations of pyruvate inhibit the formation of both AMC and a-acetolactic acid. I&ntijicatim

of AMC Produced by Ascaris

In an attempt to isolate sufhcient quantities of AMC for a more precise identification by the formation of a derivative, four 125 ml Warburg vesselseach with 50 ml of fermentation mixture were shaken for three hours at 37” under an atmosphere of nitrogen. Each 50 ml of fermentation mixture contained 10 ml of homogenate, 7.5 mg of diphosphothiamine, 0.13 millimoles of MnClz, 5.0 milliioles of sodium pyruvate and 4.0 millimoles of sodium acetate buffer, pH = 5.6. At the termination of the incubation period, the contents of the four flasks were pooled and deproteinized with NaOH and ZnSOd according to Somogyi (1930). The precipitate was washed with 50 ml of water and the washings were added to the supernate. The combined supernates contained

486

SAG,

VIDRINE

AND

HUBBARD

17.6 mg of AMC as determined by the calorimetric method of Westerfeld (1945). The AMC was then oxidized quantitatively to diacetyl with FeC13 in the presence of H&O4 (Stotz and Raborg, 1943). The diacetyl was concentrated to a final volume of 2 ml by repeated distillation and converted to 2,3-dimethyld , 6-benzoquinoxaline as described by Berl and Bueding (1951). After one recrystallization from 50% ethanol, white needles were obtained which melted at 101-102”. This is the same melting point as reported by Berl and Bueding. The melting point remained unchanged after mixing this compound with the benzoquinoxaline derivative of diacetyl. AnaZyai-ClrNnHlz (samples dried in vucuo at room temperature). Calculated Found, derivative of compound produced by Ascaris

C 80.74, H 5.81, N 13.45 C 80.80, H 5.78, N 13.44

Determination of the Optical Rotation of AMC Produced by Ascaris

In order to determine the optical rotation of the AMC produced by Ascaris, a relatively large quantity of the compound was necessary. For this purpose, 200 ml of fermentation mixture with both pyruvate and acetaldehyde as substrates were incubated in four 125 ml Warburg vessels. The contents of each vessel were as follows: 10 ml of homogenate, 7.5 mg of diphosphothiamine, 0.13 millimoles of MnClz , 5.0 millimoles of sodium acetate, 1.2 millimoles of acetaldehyde and 4.0 millimoles of sodium acetate buffer (pH = 5.6). The total volume was 50 ml. The vessels were incubated, deproteinized and the residue washed as described in the previous experiment. Then, 3.5 g of 5,5dimethyl-l ,3-cyclohexanedione (dimedon) was added to the supernate to fix the remaining acetaldehyde. The solution was then filtered and distilled in vaeuo (22-29’; 15 mm) and the distillate was collected in a dry ice-acetone trap. The first 7 ml of distillate were discarded to insure the absence of acetaldehyde. Sodium bisulfite was added to the distillate and the AMC-bisulfite complex was concentrated by distilling in vucuo and the AMC recovered as described by Berl and Bueding (1951). The final concentration of AMC was 3.05 mg per ml [alo = -79”.

ACETYLMETHYLCARBINOL

FORMATION

BY ASCARIS

487

DISCUSSION

Ascaris lumbricoides

muscle is capable of catalyzing the enzymatic synthesis of ol-acetolactic acid and of AMC from pyruvic acid. Since no a-acetolactic acid decarboxylase was detectable in Ascaris muscle, a-acetolactic acid is probably not a precursor of AMC. It appears to be more likely that AMC is formed by an acyloin condensation reaction of pyruvic oxidase (Juni and Heym, 1956). Presumably “active” acetaldehyde is formed upon decarboxylation of pyruvic acid. This ‘
488

SAZ,

VIDRINE

AND

HUBBARD

Therefore, a-acetolactic acid may be a precursor of tiglic and ar-methylbutyric acids as illustrated in the following sequence of reactions. CB;

CH,

I c=o

I

I

-

DPNH

H&--&OH I COOH a-Acetolactic acid

C&

H-C-OH I H&--&OH I COOH

DPNH ___f

H&--&-H I COOH a-Methyl-acetoacetic acid

CHa

&Hydroxya-ethylbutyric acid

c=o I

-

~+Dihydroxy-amethylbutyric acid

H-C-OH I HI C-C-H l COOH

I -

-H,O

CHa

CB[r I -

-HgO

“-;I: Ha C-C I COOH Tiglic acid

-2H

&I

8

;

-I COOH a-Methylbutyric acid

Evidence in support of such a mechanism for fatty acid formation has recently been obtained by Saz and Hubbard (1957b), who have isolated and partially purified an extract from Ascaris muscle. This system catalyzes the reduction of cu-acetolactic acid by reduced diphosphopyridine nucleotide (DPNH). In addition it catalyzes the reduction of cu-methylacetoacetic acid by DPNH. This enzyme system appears to be specific for a-methylacetoacetic acid and has been obtained free of acetoacetic acid reductase activity. In the crude Ascaris muscle homogenate, the rates of reduction of both a-acetolactic acid and cr-methylacetoacetic acid are of the same order of magnitude as the activities of most of the glycolytic enzymes, the “malic” enzyme and succinic dehydrogenase. Both systems are far more active than the lactic dehydrogenase of Ascaris muscle. Although a wide range of values for the specific rotation of ( - )acetylmethylcarbinol has appeared in the literature much of the confusion results from the methods employed in the isolation and concentration of the AMC. The specific rotation of -79” for the As-cam’s system is in good agreement with the findings of -82” for bacterial

ACETYLMETHYLCARBINOL

FORMATION

BY ASCARIS

489

systems and -84” for the Litomosoides carinii system (Berl and Bueding, 1951). According to Berl and Bueding (1951), the methods used in this investigation for the concentration of AMC entail a loss in the optical activity of 8 %. On this basis, the corrected [oL]=would be -85”. SUMMARY

1. Ascaris lumbricoides var. suis has been shown to produce acetylmethylcarbinol (AMC) as a normal product of its metabolism. Pyruvate, acetaldehyde, and pyruvate plus acetaldehyde stimulate AMC formation. 2. Homogenates of Ascaris muscle have been obtained which form AMC from pyruvate, acetaldehyde, or pyruvate plus acetaldehyde. In both the intact helminths and homogenates, approximately twice as much AMC is formed from acetaldehyde as from pyruvate. When pyruvate plus acetaldehyde were employed as substrates, AMC formation was stimulated 8- to lo-fold. 3. Homogenates also catalyze the conversion of pyruvate to cu-acetolactic acid which accumulates in the reaction. Attempts to demonstrate an ar-acetolactic acid decarboxylase have failed. 4. The corrected specific optical rotation of the enzymatically formed AMC was -85”. REFERENCES E., AND SCHIEFFELIN, W. J. 1889. Ueber Hydrirung von Ortho- und Paranaphtylendiamin und tiber 2,7-Naphtylendiamin. Ber. them. Ges. 22, 1374-1384. BERL, S., AND BUEDING, E. 1951. Metabolism of acetylmethylcarbinol in filariae. J. Biol. Chem. 191, 491418. BUEDING, E. 1953. Formation of tiglic and n-valeric acids by bacteria-free Ascaris lumbricoides. J. Biol. Chem. 202, 595-512. BUEDING, E. 1957. Personal communication. BUEDING, E., AND CHARMS, B. 1952. Cytochrome c, cytochrome oxidase, and succinoxidase activities of helminths. J. Biol. Chem. 196, 615627. BUEDING, E., AND YALE, H. W. 1951. The production of a-methylbutyric acid by bacteria-free Ascaris lumbricoides. J. Biol. Chem. 193, 411423. DOLIN, M. I., AND GUNSALUS, I. C. 1951. Pyruvic acid metabolism II. An acetoinforming enzyme system in Streptococcus faecalis. J. Bad. 62, 199-214. EPPS, W., Weiner, M., AND BUEDING, E. 1959. Production of steam volatile acids by bacteria-free Ascaris lumbricoides. J. Znfectioua Diseases 87, 149-151. GREEN, D. E., WESTERFELD, W. W., VENNEBLAND, B., AND KNOX, W. E. 1942. Carboxylases of animal tissues. J. Biol. Chem. 146. 69-84. JUNI, E. 1952. Mechanisms of formation of acetoin by bacteria. J. Biol. Chem. 196, 715-726. BAMBERGER,

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SAZ,

VIDRINE

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HUBBARD

E. AND HEYM, G. A. 1966. Acyloin condensation reactions of pyruvic oxidase. J. Biol. Chem. 218, 365-378. KEAMPITZ, L. 0. 1948. Synthesis of a-acetolactic acid. Arch. Biochem. 17,81-85. LASER, H. 1944. The oxidative metabolism of Ascaris suis. Biochem. J. 38,333-338. SAZ, H. J., AND HUBBARD, J. A. 1957a. The oxidative decarboxylation of malate by Ascaris lumbricoides. J. Biol. Chem. 226, 921-933. SAZ, H. J., AND HUBBARD, J. A. 1957b. Unpublished observations. SOMOQYI, M. 1930. A method for the preparation of blood filtrates for the determination of sugar. J. Biol. Chem. 88, 6.55463. STOTZ, E., AND RABORQ, J. 1943. A calorimetric determination of acetoin and diacetyl. J. Biol. Chem. 169, 25-31. WESTERFELD, W. W. 1945. A colorimetrio determination of blood acetoin. J. Biol. Chem. 161, 495-502. JUNI,