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ANALYTICAL BIOCHEM ISTRY 17, 369-376 (1966)

Determination of Citrate with Citrate LyaseHANS MOELLERING AND WOLFGANG GRUBER

BiochenG cal Division Tutzing/Obb., C. F. Boehringer & Soehne Gmbli,Mannheim, GeTmany

Received June 1, 1965

Citric acid is known to be an important intermediate in metabolism.Accordingly, it is found widely in plant and animal material. A rapid,convenient micromethod of determination would be of interest to bio-chemists and food chemists as well. Some methods exist: The colori-metric determination after conversion to pentabromoacetone is mostcommonly used ; interfering substances like acetone, acetaldehyde or,Q-keto acids must be previously removed (1). The enzymic method ofestimation using aconitase and isocitric dehydrogenase (2) is of limitedvalue because of the low stability of the aconitase preparations. How-ever, enzymic cleavage of citrate with citrate lyase (citrate oxaloacetate-lyase, EC 4.1.3.6) and determination of the resulting oxaloacetate by themalate dehydrogenase reaction appears to be promising:

citrate lyasecitrate - OX + acetate il)RIDHOA + NADH + H+ - malate + NAD+ , j

A procedure utilizing citrate lyase was published by Dagley (3). How-ever, this method, which uses magnesium ions as cofactor, suffers fromthe limitation that a given amount of citrate lyase will catalyze thedecomposition of only a limited amount of citrate before the enzymebecomes inactive (4-11). The linear relationship between optical densitychanges and the initial citrate concentration of the sample is thereforevalid only at very low concentrations. It was found by Dagley andDawes (5) with high concentrations of citr:ite that zinc ions at low con-centrations are nearly as good activators for citrate lyaee as magnesiumions. At concentrations above 1 mnd Zn++ activation is still better, so thata greater amount of citrate can be decomposed by a given amount of

1 Abbreviations used: KA D, NADH = oxidized and reduced forms of tlipllus-phopyridine nuclco tide ; OA = osaloa cctate ; Py = pyruvate ; MDH =J lnn licdehydrogenase ; LDH = lacti c dehydrogenase; ADH = alcoho l dchydrogenase ; CLZZ citrate lyase.

369

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370 MOELLERING AND GRUBERenzyme before inactivation occurs. We have found (12) that the higheractivity of citrate lyase in the presence of zinc ions is due to a stabiliza-tion of the enzyme. On incubation with 0.130 m&I citrate and varyingamounts of zinc ions, the speed of inactivation is related inversely tothe molar ratio of zinc to citrate with optimum stability at the ratiosl-4. Under these optimal conditions the enzyme lost only 30-50s of itsactivity within 10 min. In analogous experiments with magnesium ionsmore than 95 inactivation occurs irrespective of the concentration ofmagnesium present or the magnesium:citrate molar ratio.Since the most inhibitory substance is a complex of the enol trianion(7) of oxaloacetate with magnesium ions (4, 5, 10, ll), we performedexperiments in which citrate lyase was incubated with various concen-trations of oxaloacetate and zinc ions. The inactivation profiles wefound were very similar to those of the incubations with equal concen-trations of citrate and zinc ions. We assume that zinc ions react withoxaloacetate differently than magnesium ions and that the zinc complexis less inhibitory. This assumption is supported by differences in thespectral shifts caused by zinc ions on oxaloacetate compared with thosecaused by magnesium ions (12).The procedure of Dagley (3) is based on the fact that the oxaloacetateformed from citrate is decomposed to pyruvate by oxaloacetate de-carboxylase (EC 4.1.1.3) present in cell-free extracts. In our procedurewe eliminate the uncertainty that sufficient amounts of the decarboxylasemay not be present for this conversion by using a mixture of lactatedehydrogenase (EC 1.1.1.27) and malate dehydrogenase (EC 1.1.1.37).Any pyruvate that is formed from oxaloacetate by a nonenzymic reac-tion is assayed by the first enzyme, while unchanged oxaloacetate isassayed by the second. By the coupling with the dehydrogenase systemsthe equilibrium of the citrate lyase reaction, K = 3.08 (lo), is shiftedto the direction of complete splitting of citrate. Prior to the determina-tion of citrate, pyruvate and oxaloacetate can be assayed in the samemixture.

METHODSReagents

All solutions were made up with glass-distilled water.I. Buffer: 0.1 M triethanolamine, pH 7.6.II. Ca. 0.01 M /3-NADH.III. Ca. 0.03 M zinc chloride.IV. 0.6 M perchloric acid.V. 0.1 M citric acid, pH adjusted to 7.5 with NaOH.

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DETERMINATION OF CITRAT E 371VI. Lactic dehydrogenase, LDH, 2 mg protein/ml (360 IU/mg).VII. Malic dehydrogenase, MDH, 2 mg protein/ml (720 IU/mg) .VIII. Citrate lyase, ca. 10 mg protein/ml, ca. 10 IU/mg. The enzymewas partially purified in a similar way to tliat described by Dagely (3)or Tate and Datta (10) from Aerobacter aerogcn.es. t was stabilized byaddition of a combination of bovine serum albumine, sucrose, and mag-nesium sulfate and lyophilized (see note 1).Zinc chloride, perchloric acid and citric acid were obtained from E.Merck, Darmstadt; all other reagents used were Biochemica Boeb-

ringer.Stability of Reagents: The NADH solution can be stored for 3 weeksat 0C in the dark. The LDH and MDH suspensions are stable forseveral months when kept at 0. The citrate Iyase loses ca. 10% of itsactivity within 1 month when stored in the cold.

Procedure for Activity Determinations of Citrate LyaseWavelength 366 nm; glass cuvet of 1 cm light path; temperature 25C.The experimental measurements were made against air with anEppendorf photometer; however, other spectrophotometers may be used.The reagents are pipetted into the cuvet in the following order:

Reagent Final concentration2.66 ml buffer (I) 95 mM0.06 ml NADH solution II) 0.2 mM0.20 ml ZnClt solution (III) 2.0 mf if0.05 ml citrate solution (V) 1.7 mM0.01 ml LDH suspension (VI) 2 IU LDH/ml0.01 ml MDH suspension (VII) 5 IU XIDH/ml

Then 0.01 ml citrate lyase suspension (VIII) is mixed in.After the optical density has decreased by 0.020, the time required fora further decrease in optical density of exactly 0.050 is measured. The

specific activity is calculated by the formula:difference in extinction X assay volume unitsmeasured time (min) X mg enzyme taken X mhl extinction of NhDH = mg

Procedure for Determination of CitrateMaterials: Citrate can be determined in wine, fruit juices and othersamples with low protein content without any preliminary treatment.Samples from animal tissue, serum, and extracts containing protein mustbe deproteinized prior to the assay. Deproteinization must be performedwith reagents which do not form precipitates with zinc ions. Perchloricacid was found to be suitable.Deproteinization: 5 gm of tissue is homogenized immediately after

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372 MOELLERING AND GRTJBERremoval with 10 ml of precooled 0.6 M perchloric acid (IV) in the cold.After centrifugation, 11 ml of supernatant is neutralized with 2 ml of2 N KOH and kept in an ice bath for 15 min. The precipitated KClO, isfiltered off,Assay: Wavelength 366 nm, glass cuvet of 1 cm light path, roomtemperature. Measurements are made against air with an Eppendorfphotometer; however, other spectrophotometers may be used.The reagents are pipetted into the cuvet in the following order:

Reagent2.00 ml buffer (I)0.06 ml NADH solution (II)0.01 ml ZnCl, solution (III)0.02-0.9 ml sample (fruit juices 0.02 ml, wine 0.2 ml,deproteinized tissue samples 0.9 ml)

Final concentration67 mM0.2 mM0.1 mM

Water is added to obtain a volume of 2.97 ml and El is read; then 0.01ml LDH suspension (V) and 0.01 ml MDH suspension (VI) are mixedin.

Through the action of these enzymes the pyruvate (Py) and oxalo-acetate (OA) present in the sample are removed. The end point in opticaldensity E, is reached within 3 min. From EPy+OA E, - E,, one cancalculate the total amount of pyruvate + oxaloacetate present. The con-centration of each individual compound can be measured by separateaddition of LDH and MDH.

Then 0.01 ml citrate lyase suspension (VIII) is mixed in. Citrate isdecomposed within 5-10 min, and the E, is read. The difference in opticaldensity due to citrate is:AE,it, = Ez - Ez

The amount of citrate is calculated from the formula:AE,it, X 0.174 mg citric acid=

V ml samplesince

AEVh mg substance---Zcdl/ ml samplewhere E = optical density coefficient of NADH = 3.3 X lo3 cm2/mmoleat 366 nmd = light path of cuvet = 1 cmV = assay volume = 3 mlZJ= sample volume = 0.02-0.9 ml, andM = molecular weight = 192.1.

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DETERMINATION OF CITRAT E 3i3

LDH/ MDH

/Ext. /CL366nm

f0.500

0.400

0.300

0.200

0.100

I I L I I I,, 1 I, I I I I0 5 10 15-b t [min]

FIG. 1. Determination of citrate in wine (0.4 ml). For details see text.

Figure 1 shows the changes in extinction that occurred during thedetermination of citrate in wine.NOTES

1. Purity of Citrate LyaseOur purified preparations of citrate lyase are practically free (

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374 MOELLERING AND GRUBERcitrate lyasecitrate - oxaloacetate + acetate OA decarboxylase +

Py decarboxylasepyruvate + COZ ) acetaldehyde f COP (3)ADHacetaldehyde + NADH + H+ - ethanol f NAD+ (4)

2. Determination of CitrateUnder the conditions given earlier, the citrate cleavage reaches com-pletion within 5-10 min. The amount of Zn++ added to the assay mixturemust be limited in the presence of phosphate or carbonate in the samples

to avoid precipitation. In these cases, the concentration of citrate lyaseshould be increased by the same factor by which Zn++ is reduced. Quan-tities of citrate as low as 0.02 pmole (corresponding to 0.004 mg citricacid) may be assayed with satisfying accuracy (+50/O). The sensitivityof the method may be increased further by measuring the consumptionof NADH at 340 nm or by fluorometric estimation.

3. Accuracy of the MethodThe accuracy of the method is shown in Table 1, which shows the re-covery of citrate found on addition of increasing amounts to a depro-teinized homogenate of rabbit liver.

TABLE 1Recoveries of Citrate Added to Rabbit Liver HomogenateCitrate added EdI

1 ml homogenate 0.040+ 0.03 0.074+ 0.06 0.110+ 0.09 0.134+0.12 0.170+ 0.15 0.202

Citrate found,WlOk

0.0364 f 0.00070.06730.10100.12240.15440.1844

70 Recov.

-1031079698.599

To improve the accuracy of the method, the citrate content of thehomogenate lacking citrate (Table 1) was determined 10 times. Theerrors lay within limits of -2 and 41.5 . In wine, which has a highercitrate content, the maximal error was &lPr,.

4. Specificity of Citrate LyaseThe enzyme is absolutely specific for citrate. We examined isocitrate,&.s-aconitate, oxalate, succinate, fumarate, a-ketoglutarate, n-glutamate,tartrate, lactate, malate, acetate, ascorbate, glucose, fructose, and eth-anol at concentrations similar to citrate. None of these substances reacted

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DETERMINATION OF CITRAT E 315or exhibited any interference with speed and accuracy of the determina-tion of citrate by this method.

5. Application of the MethodThe method was applied to various materials of animal and plant

origin. Table 2 gives our results.TABLE 2

Citrate Content of Biolo gical Materials and of FoodsSubstrate Citric acid content

Human serumRat liverRat heartRabbit liverWine (Nahe)Cherry juice (Vitaborn)Apricot juice Blueberry juice ICurrant juic e Orange juic e (fresh)Lemon juice (fresh)Frankfurter sausages

0.022 mg/ml0.024 mg/gm fresh wt.0.021 mg/gm fresh wt.0.021 mg/gm fresh wt.0.200 mg/ml0.172 mgjml4.13 mg/ml4.48 mg/ml7.00 mg/ml

13.9 mg/ml65.X mg/ml

1.3 mg/gm wt.

SUMMARYA method is described for the rapid determination of citrate in bio-

logical material which is based on the complete cleavage of the sub-stance by purified preparations of citrate lyaze in the presence of zincions. The assay of citrate lyase is also markedly improved by additionof zinc. The method was applied to a number of animal tissues andfruit juices. It is absolutely specific for citrate.

ACKNOWLEDGMENTSTh e authors are greatly indeb ted to Dr. H. U. Brrgmeycr for many help ful dis-

cu ssion s and to Dr. G. Holz for the culture of npproprixte strain s of Aerohn cieraerogenes.

REFERENCES1. STERN, J. in S. P. Colowick u. N. 0. Kaplan &Ict.hods in Enzymology, Vol. 3,

p. 426. Acad emic Press, New York, 1955.2. SIEBERT, G. in H. U. Bergmeycr Methods of Enzym atic An alys is, p. 318. Icrlag

Chcm ie Weinhe im and Acad emic Press, Ncs\v l-ark, 1963.3. DAGLET, S. in H. U. Bergmeyer Methods of l&z?-m atic An alys is, p. 313. Vcrln g

Chcm ie Weinhe im and Acad emic Press, New York, 1963.4. BOWEN, T. J. AND L. J. ROGERS, Biochem. Biophys. Acta 77, 685 (1963).5. DAGI,~ S. AXD E. A. DAWES, Bioch~nz. Uiopl~v.s. Acln 17, 177 (1955).

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376 MOELLERING AND GRUBER6. DARON, H. H., AND GUNSALUS, I. C., in Methods in Enzymology (Colowick,

S. P., and Kaplan, N. O., eds.), Vol. V, p. 622. Academic Press, New York,1962.7. EISENTHAL, R., S. S. TATE AND S. P. DATTA, 3.FEBS-Meeting, Warsaw 1966,Abstract F169, p. 264. Academic Press, New York, 1966.8. HARVEY, R. J. AND E. B. COLLINS, J. Biol. Chem. 238, 2648 (1963).9. TAT E, S. S. AND S. P. DAT TA, Biochem. J. 91, 18C (1964).10. TAT E, S. S. AND S. DAT TA, Biochem. J. 94, 470 (1965).

11. WHEAT, R. W. AND S. J. AJL, J. Biol. Chem. 217, 909 (1955).12. GRUBER, W. AND H. MOELLERING, Bioc hem . 2. 346, 85 (1966)