anion exchange

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ANION EXCHANGE CHROMATOGRAPHY OF ACIDS OFTHE CITRIC ACID CYCLEBY HARRIS BUSCH,* ROBERT B. HURLBERT, AND VAN R. POTTER

(From the McArdle Memorial Laboratory, Medical Scho ol, University of Wis con sin,Madison, Wisconsin)

(Received for publication, January 7, 1952)The demonstration of the accumulation of citrate in vivo in the presenceof fluoroacetate and the specific modification of this effect produced in

various organs by the injection of malonate (14) have increased thedesirability of isolation and quantitative determination of the acids ofthe Krebs cycle and related substances. Further, proposed studies in-volving the use of radioactive tracers require the isolation of these acidsin pure form. Although partition chromatography by means of silica gelcolumns has been used with notable success or separation of some of theseacids (5-8), difficulties in the separation of certain pairs of acids, such aslactic and succinic, and limitations on the quantities of acids resolvable,led us to the use of ion exchange chromatography (9). While ion exchangeresins were used for separation of the acidic group of amino acids from theneutral and basic groups (10, ll), the use of these resins for chromato-graphic separation of individual amino acids was first reported by Steinand Moore. (12, 13). Recently, Cohn has described the separation ofnucleotides and purine and pyrimidine bases by ion exchange columns(14). This study is-an extension of the use of these resins to the separa-tion of acids of the Krebs cycle.Materials and MethodsDowex I (Formate Form)-The resin was obtained from the manufac-turer in the chloride form. The finer particles of the 300 to 500 meshmixture were removed by discarding the supernatant after centrifugationof an aqueous suspension at 500 r.p.m., or by decantation of the finerparticles after the resin had been suspended n water and the bulk of thecoarse particles had settled. A batch of resin (600 ml. of resin bed) suf-ficient for the preparation of approximately 50 columns for analytical use

was poured into a large glass column fitted with a sintered glass disk nearthe bottom; this disk was covered with glass wool. A solution of sodiumformate (1 M) was passed through the resin bed until no further turbidity* Postdoctoral Research Fellow of the National Cancer Institute, United States

Public Health Service.1 The Dow Chemical Company, Midland, Michigan.717

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718 CHROMATOGRAPHY OF CITRIC ACID CYCLEdeveloped in the effluent on addition of an acidic solution of silver nitrate.Approximately 1 liter of sodium formate solution was required for each25 ml. of resin bed for conversion to the formate form. After the resinwas washed with distilled water, it was stirred in a small aqueous volumeto make a thick slurry, which was then poured into a storage flask.

Silica Gel-The gel was prepared by methods previously described (5, 7).For separation of malonic, citric, and isocitric acids, columns containing1 gm. of gel were developed with 35 per cent butanol-chloroform afteraddition of the sample (7).

Acids-Pyruvic acid was supplied by Dr. G. A. LePage; Dr. R. H. Burrisprovided a sample of cis-aconitic acid; dimethylisocitric la&one was fur-nished by Dr. Alton Meister; a-ketoglutaric acid had been prepared byDr. W. W. Ackermann; and oxalacetic acid had been prepared by Dr.Charles Heidelberger. The other acids used were commercial prepara-tions of highest purity available.

Apparatus-To obtain a uniform gradual increase in acidity in the resinbed, an apparatus was used which consisted of a reservoir of concentratedformic acid, a mixing flask, and a glass column containing the resin. Dropsof eluate from the resin column were collected in large test-tubes via theTechnicon automatic fraction collector,2 generally set to collect 40 drops(approximately 2.0 ml.) per tube. A controlled air inlet, equipped with amanostat3 and an air filter, admitted air to the system at a pressure suf-ficient to force fluid from the reservoir, through the mixing flask, and finallythrough the resin column. The reservoir was a 500 ml. filter flask contain-ing 6 N formic acid. The mixing flask was a 200 ml. round bottom flask,initially containing 200 ml. of distilled water. To insure thorough mixingof the concentrated acid entering the mixing flask and the dilute aqueoussolution within, a magnetic stirring bar, encased in acid-resistant plastic,was placed within the flask and spun at a rapid rate under the influence ofa rotating magnetic field.4 The resin column was prepared by adding aslurry of resin to a glass column 1 cm. in diameter, previously fitted with aremovable sintered glass disk and above the glass disk a circular paperdisk crimped up around the edges to prevent escape of the resin. Afterthe resin settled, a pledget of glass wool was placed above the column. Theheight of the resin column was 11.5 cm. and the column contained 9.1 ml.of resin bed. After the sample and wash solution had been forced intothe resin bed, 15 ml. of distilled water were added to the glass columnabove the resin bed and the column was then set in place in the apparat,us.The small volume of water above the resin bed provided a second volume

2 Tec hnico n Chromatography Corporation, 215 E. 149th Street, New York, 51.3 Moore Products Company, II and Lycoming Streets, P hiladelp hia, 24.4 Arthur H. Thom as Company, Philad elphia.

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720 CHROMATOGRAPHY OF CITRIC ACID CYCLEwash solution was also forced into the resin bed. The neutral effluentemerging from this init ial procedure was collected separately. Water wasadded above the resin bed and the column was then placed in operation asdescribed above.

Determinations-Citrate (17), lactate (18) and pyruvate and ar-keto-glutarate (19) were determined by standard methods. Solid acids weredetermined by titration with 0.01 N NaOH.

ResultsEmergence of Acid Peaks-After the positions of the peaks of individualacids had been determined, a mixture of known acids of the Krebs cycle

ANION-EXCHANGE

0d&ION N%BER 150

FICA 1and the closely related amino acids, glutamic and aspartic, was subjectedto the procedure. The chromatogram is illustrated in Fig. 1. The aminoacids were eluted first, and in a single band. These were followed by aband of lactic acid generally emerging in two or three fractions. Succinicacid emerged several fractions after the last of the lactic acid and wasfollowed by malic acid. Fractions 50 to 65 contained a mixture of pyruvic,malonic, citric, and isocitric acids, and the position of this group is ap-proximated by pyruvic in Fig. 1. The fumaric acid peak emerged in Frac-tions 80 to 95, followed by cr-ketoglutaric acid in Fractions 105 to 120 andfinally cis-aconitic acid, which appeared as a long low peak.

To separate the acids found in Fractions 50 to 65, the tubes containingthe acid eluate were each treated with 1 ml. of 2,4dinitrophenylhydrazineand then incubated for 10 minutes at 28. After two extractions withethyl ether, the entire procedure was repeated; the phenylhydrazone ofpyruvic acid and most of the phenylhydrazine were removed by the ether.

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H. BUSCH, R. B. HURLBERT, AND V. R. PO TTER 721These tubes, along with all others collected, were subjected to desiccation.Solid citric and malonic acids, isocitric lactone, and traces of phenylhy-drazone remained in the test-tubes after desiccation. After the aciditywas titrated, excess alkali was added and the solutions were placed in aboiling water bath for 1 hour to hydrolyze the isocitric lactone. Inasmuchas isocitric lactone and malonic acid are not separated by the silica gelcolumn under the conditions utilized, it was necessary to convert the iso-citric la&one to isocitric acid, which was separated (Fig. 2), and quantita-tively transfer the latter to the organic phase. Quantitative transfer ofthe acids to the organic phase from an acidified aqueous solution6 waseffected by the transfer technique of Marshall, Orten, and Smith (5).The mixture of acids, dissolved in 35 per cent butanol-chloroform, was

cn SILICA GELl-=4.8--I 0 9a> s3 04w2.4- ghw051 II I0 30 60FRACTION NUMBER

FIG. 2

chromatographed on a 1 gm. silica gel column, developed with 35 per centbutanol-chloroform (7). The traces of phenylhydrazone emerged in thefirst fraction collected, while the malonic, citric, and isocitric acids emergedas shown in Fig. 2.

Recovery-Table I presents data on recovery of acids when added to theanion column singly or in mixtures. Recovery of acids was not signifi-cantly different when a mixture of acids was carried through the entireprocedure (Table II, Experiment A). There was no change in the posi-tions or recovery of the emergent acids when a mixture was made 0.33 Nwith respect to perchloric acid and the perchlorate was precipitated as thepotassium salt in accordance with the procedure presented under Prepa-ration of tissue samples (Table II, Experiment B). Although most acidswere determined by titration, lactate and pyruvate were determined colori-

6 Considerable formation of the lactone (50 to 75 per cent) was found when thesalts were dried in the presence of excess HCI or when the free a cid was dried afterpassag e through a cation exchange colum n in the hydrogen form.

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722 CHROMATOGRAPHY OF CITRIC ACID CYCLETABLE I

Recovery of Ac ids from Anion Exchange ResinAc ids were added to the res in column in a range of 5 to 200 MM. individually or

in small groups. I

Acid No. ofexperiments

Glutamic .........Lactic. ............Succinic ..........Malic .............Malonic .Citric. ............Pyruvic. ..........Isocritic. ..........Fumaric. .cu-Ketoglutaric..cis-Aconitic.

.

I 25877635443/ I

TABLE II

I- verage Averagerecovery deviationgercent10291969295949097855233

per cent2457836827

Rangeper cent98-10689- 9480-10282- 9886-11182-11187- 9483-10372-10050- 5623- 38

Recovery of Ac ids after Addition of Known Mixtures to Anion Exchange ResinThe total procedure include d separation of pyruvic acid as the 2,4-dinitrophenyl-

hydrazone and resolution of citric, m alonic, and isoc itric acid s by chromatographyon sili ca gel colum ns. In Experiment B, the mixture of acid s was treated withperchloric acid in the same concentration as noted in Preparation of tissue samp lesand the procedure was carried on from that p oint.

I Experiment AAcidAdded I 2ecoverec

PMGlutamic and aspartic. .......Lactic ........................ 10Su cc ini c. ..................... 50Malic. ....................... 100Malonic ..................... 50Pyruvic. ..................... 50Citric. ....................... 100Isocitric ......................Fum aric ...................... 100cr-Ke toglutaric. ............... 100cis-Aconitic. .................

9.0 90.050.0 100.082.5 82.546.0 92.047.0 94.095.5 95.5

84.0 84.053.0 53.0

per cen

Experiment B

-- Added R ecovered RW%lyPM PM per tent50 49.0 98.010 9.2 92.015 14.5 96.320 17.3 87.015 12.9 86.010 8.9 89.010 9.1 91.0

8 7.6 95.020 20.0 100.030 16.8 56.0

100 38.0 38.0

metrically; in each of these cases, there was interference by the formicacid eluent or traces of formaldehyde in it, and appropriate correctionsfor these effects have been made in Tables I to III. Inasmuch as removalof formic acid by desiccation is practically quantitative, the blank valuefor titration was very low, generally less han 0.1 microequivalent of base.

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H. BUSCH, R. B. HURLBERT, AND V. R. PO TTE R 723Considerable losses of a-ketoglutaric and cis-aconitic acids were ex-

perienced: calorimetric analysis of the a-ketoglutarate fraction before desic-cation revealed the same quantity of acid to be present as was found bytitration. Low recoveries of cis-aconitate were obtained under the stand-ard desiccation conditions noted above, as compared with desiccation inthe absence of heat; i.e., more than 90 per cent of the cis-aconitic acid waslost under the former conditions. Recoveries greater than 40 per cent havenot yet been achieved. Lactic and pyruvic acids were quantitativelyremoved by the desiccation procedure employed. Calorimetric analysesof the formic acid eluate revealed that these acids emerged from thecolumn quantitatively.

TABLE IIIReproduc ibility of Pos ition of Emergent Acid . Peaks from Anion Exchange Resin

Summary of experiments in which known mixtures were added to the resincolumns.

Acid I Fraction Nos.Experimen19 t1 t 1xperimen23 hperimenl t125 Cxperiment31SuccinicLactic.Malic.MalonicCitric. .PyruvicFumaric.a-Ketoglutaric.

26 301733- 366Q- 69

84- 9312(f126

28-3235-39667253-6186-91

24r 2815- 1930- 37

53- 6387-100114-134

29- 3236- 406s 71

89- 99114-129

: E-

kperimen34 tE kperiment423&32 26- 29IQ-20 1% 2036-40 33- 366G-71

56- 6380- 90108-119

Identijication of Emergent Acids-Citric and lactic acids were identif iedby calorimetric analysis, while the identity of pyruvic and a-ketoglutaricacids was established by spectrophotometric study of the phenylhydra-zones. Melting points of the solid residue in the succinic, malic, malonic,and fumaric acid fractions were 188, 99, 132, and 286 respectively,compared to 189, 100.5, 133, and 290 for authentic samples. Theother acids were identified by the position of the peak and the acid equiva-lence of micromoles added and micromoles recovered. Citric and iso-citric acids presented only two titratable acid groups per molecule afteremergence from the anion exchange column; the latter formed a lactonewhich was hydrolyzed by heating in the presence of excess alkali. Re-producibility of the positions of the peaks of known acids added to thecolumn is indicated in Table III.

Isolation of Acids from Tissues Following Injection of Inhibitors in Vivo-

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724 CHROMATOGRAPHY OF CITRIC ACID CYCLEAs an illustration of the use of this method for analysis of tissue samples,the citrate content of livers and kidneys of rats treated with fluoroacetatewas determined by calorimetric analysis of the supernatant solution afterthe homogenates were treated with perchloric acid and was compared withcitrate found after passage through the column. Recovery of the citratewas determined both by calorimetric analysis and by titration; between90 and 100 per cent of this acid was recovered. Further experiments werethen undertaken which involved estimation of al l the acidic components

PEAK I 2 3 4 5 6 7CONTROL

20-

IwJ 30- FLUOROACETATE2 R

RLI / Iz 30- MALONATE

n20-

I I0 40 80 I:FRACTION NUMBER0

FIG. 3emerging in the range of the chromatogram of the acids of the citric acidcycle.In Fig. 3, titration of acidity in the fractions collected is plotted againstthe number of the fraction for representative experiments on normal kid-neys (control), kidneys of rats treated with fluoroacetate, and kidneys ofrats treated with malonate. Such graphs may be referred to as the acidprofile of the sample. The acid profile of normal kidney contained sevenwell defined peaks of acidity. Peaks 1 and 2 contained substances pro-ducing a positive ninhydrin test and Peak 2 was in the same position asglutamic and aspartic acids. Peak 4 contained traces of a dark brown oilafter desiccation, while Peak 7 contained inorganic phosphate. Furtherinvestigation of these peaks is in progress. In the fluoroacetate study, a

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H. BUSCH, R. B. HURLBERT, AND V. R. PO TTER 725marked increase in titration was found in the peak which contains thecitrate fraction (Peak 6), and the citrate content by titration correlatedwell with the citrate content as determined by calorimetric analysis. Inthe acid profile of the kidney of the rat treated with malonate, increasedacidity was found in Peak 4, which corresponds to the succinate peak inchromatograms of known acids, as well as in Peak 6, which correspondsto the malonate peak in chromatograms of known acids. The succinatepeak was contaminated by the brown oil of Peak 4, as was apparent bothby gross inspection and depression of the melting point of the crystals ofsuccinic acid from 189 to 165. From these preliminary experiments,it was evident that the acid profile mirrored changes in a number of acidmetabolites and provided a basis for further purification of the componentsof the peaks. Inasmuch as a number of substances could be presentin any given peak of the acid profile, the use of inhibitors permitted theaccumulation of a large quantity of the component acid desired, andchromatography of a small sample reduced the content of the other com-ponents to negligible quantities.

DISCUSSIONIt is apparent from the data presented that the position of the acids in

the eluate is dependent upon a number of factors which include the numberof carboxyl groups and the ionization constant. The r81e of the numberof carboxyl groups is indicated in the following examples. The ionizationconstant of the most active carboxyl group in pyruvic and cr-ketoglutaricacids is essentially the same; yet the former emerges some 50 fractionsearlier than the latter. Moreover, the first ionization constant of malonicacid is half that of pyruvic acid and twice the first ionization constants ofcitric or isocitric acid; yet al l emerge in the same fractions. The relativepositions of the succinic, malic, and fumaric acid peaks are evidence forthe r&e of the ionization in adherence of the acids to the resin; the sameholds true for lactic and pyruvic acids.

Anion exchange chromatography possesses several advantages over silicagel methods in the separation of acids of the Krebs cycle. First, lacticand succinic acids were completely separated; second, the blank titrationswere very low, and, in addition, valuable information was obtained by de-termination of the melting points of the crystals remaining in the tubes afterdesiccation. Finally, the capacity of the resin column is much greater.As much as 150 mg. of malonic acid and 25 mg. of succinic acid has beenrecovered from columns of the type used in this study without appreciablechange in their positions, while silica gel columns of 5 times the resin bedvolume used enable separation of 1 to 15 mg. of a mixture of acids of theKrebs cycle (6).

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726 CHROMATOGRAPHY OF CITRIC ACID CYCLEThis method lends itself to various types of studies; it has been used

successfully in Warburg experiments involving oxidation of labeled py-ruvate, as well as in studies on the effects of malonate on tumors andnormal tissues in viva. Moreover, it is useful to have a method whichcan be used in conjunction with partition chromatography of organic acids,since it is very unlikely that two substances will have the same elutioncharacteristics on both types of column.

SUMMARY1. Formic acid, in continuously increasing concentration, eluted acidsof the citric acid cycle and related acids from a Dowex 1 anion exchange

resin (formate form) in the following order: glutamic and aspartic; lactic;succinic; malic; a mixture of pyruvic, malonic, citric and isocitric; fumaric;a-ketoglutaric; cis-aconitic. The components of the mixture containingpyruvic acid were separated by extraction of the latter as the 2,4-dinitro-phenylhydrazone, followed by chromatography of malonic, citric, and iso-citric acids on silica gel columns.

2. Recovery of most acids ranged from 90 to 100 per cent, but lowrecoveries of cr-ketoglutaric and cis-aconitic acids were found. The posi-tions of individual acid peaks on the chromatograms were consistent.

3. The method was used to demonstrate an increase in the citrate con-tent of perchloric acid extracts of kidneys of rats treated with fluoroacetateand an increase in succinate and malonate content of extracts of kidneysof rats treated with malonate.

The authors wish to express their indebtedness to Dr. G. C. Muellerand Dr. G. A. LePage for their helpful suggestions in this work.BIBLIOGRAPHY

1. Buffa, I?., and Peters, R. A., Nature, 163, 914 (1949).2. Buffa, P., and Peters, R. A., J. Physio l., 110, 488 (1950).3. Potter, V. R., and Busc h, H., Cancer Res., 10, 353 (1950).4. Potter, V. R., Proc. Sot. Exp. Bio l. and Med., 76, 41 (1951).5. Marsha ll, L. M., Orten, J. M., and Sm ith, A. H., J. Biol. Chem., 179, 1127 (1949).6. Frohman, C. E., Orten, J. M., and Sm ith, A. H., J. Bio l. Chem ., 193, 277 (1951).7. Isherwood, F. A., Bioche m. J., 40, 688 (1946).8. Marvel, C. S., and Rand s, R. D., Jr., J. Am . C hem. Sot., 72, 2642 (1950).9. Bu sch , H., Hurlbert, R. B., and Potter, V. R., Federation Proc., 10, 169 (1951).

10. Cannan, R. K., J. Bio l. Chem., 162, 401 (1944).11. Cons den, R., Gordon, A. H., and Martin, A. J. P., Bio che m. J., 42, 443 (1948).12. Stein, W. H., and Moore, S., Cold Spring Harbor Sympo sia Quant. Biol., 14, 179

(1949).13. Moore, S., and Stein, W. H., J. Bio l. Chem., 192, 663 (1951).14. Cohn, W. E., J. Am. Chem. Sot., 72, 1471 (1950).

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H. BUSCH, R. B. HURLBERT, AND V. R. PO TTE R 72715. Potter, V. R., Bus ch, H., and Bothwe ll, J., Proc. Sot. Exp. Bio l. and Med., 76,

38 (1951).16. Potter, V. R., and Elvehjem, C. A., J. Bio l. Chem., 114, 495 (1936).17. Natelson, S., Lugovoy, J. K., and Pinc us, J. B., J. Bio l. Chem., 170, 597 (1947).18. LePage, G. A., in Umb reit, W. W., Burr-is, R. H., andstau ffer, J. F., Manom etric

technique s and tissue metabo lism, Minneap olis (1949).19. Friedemann, T. E., and Haugen, G. E., J. Bio l. Chem., 147,415 (1943).

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