ANALYTICAL

BIOCHEMISTRY

17,

369-376

(1966)

Determination
HANS
BiochenGcal

of Citrate
AND

with Citrate
WOLFGANG

Lyase
GRUBER
& Soehne Gmbli,

MOELLERING
Division Tutzing/Obb., Mannheim, Received

C. F. Boehringer GeTmany 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 biochemists and food chemists as well. Some methods exist: The colorimetric determination after conversion to pentabromoacetone is most interfering substances like acetone, acetaldehyde or commonly used ; ,Q-keto acids must be previously removed (1). The enzymic method of estimation using aconitase and isocitric dehydrogenase (2) is of limited value because of the low stability of the aconitase preparations. However, enzymic cleavage of citrate with citrate lyase (citrate oxaloacetatelyase, EC 4.1.3.6) and determination of the resulting oxaloacetate by the malate dehydrogenase reaction appears to be promising:
citrate lyase

citrate OA + NADH

RIDH

OX + H+ -

+ acetate malate + NAD+

il)
,"j

A procedure utilizing citrate lyase was published by Dagley (3). However, this method, which uses magnesium ions as cofactor, suffers from the limitation that a given amount of citrate lyase will catalyze the decomposition of only a limited amount of citrate before the enzyme becomes inactive (4-11). The linear relationship between optical density changes and the initial citrate concentration of the sample is therefore valid only at very low concentrations. It was found by Dagley and Dawes (5) with high concentrations of citr:ite that zinc ions at low concentrations are nearly as good activators for citrate lyaee as magnesium ions. At concentrations above 1 mnd Zn++ activation is still better, so that a greater amount of citrate can be decomposed by a given amount of
1 Abbreviations used: KAD, NADH = oxidized and reduced forms of tlipllusphopyridine nuclcotide ; OA = osaloacctate ; Py = pyruvate ; MDH =J lnnlic dehydrogenase ; LDH = lactic dehydrogenase; ADH = alcohol dchydrogenase ; CL ZZ citrate lyase. 369

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enzyme before inactivation occurs. We have found (12) that the higher activity of citrate lyase in the presence of zinc ions is due to a stabilization of the enzyme. On incubation with 0.130 m&I citrate and varying amounts of zinc ions, the speed of inactivation is related inversely to the molar ratio of zinc to citrate with optimum stability at the ratios l-4. Under these optimal conditions the enzyme lost only 30-50s of its activity within 10 min. In analogous experiments with magnesium ions more than 95% inactivation occurs irrespective of the concentration of magnesium 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 performed experiments in which citrate lyase was incubated with various concentrations of oxaloacetate and zinc ions. The inactivation profiles we found were very similar to those of the incubations with equal concentrations of citrate and zinc ions. We assume that zinc ions react with oxaloacetate differently than magnesium ions and that the zinc complex is less inhibitory. This assumption is supported by differences in the spectral shifts caused by zinc ions on oxaloacetate compared with those caused by magnesium ions (12). The procedure of Dagley (3) is based on the fact that the oxaloacetate formed from citrate is decomposed to pyruvate by oxaloacetate decarboxylase (EC 4.1.1.3) present in cell-free extracts. In our procedure we eliminate the uncertainty that sufficient amounts of the decarboxylase may not be present for this conversion by using a mixture of lactate dehydrogenase (EC 1.1.1.27) and malate dehydrogenase (EC 1.1.1.37). Any pyruvate that is formed from oxaloacetate by a nonenzymic reaction is assayed by the first enzyme, while unchanged oxaloacetate is assayed by the second. By the coupling with the dehydrogenase systems the equilibrium of the citrate lyase reaction, K = 3.08 (lo), is shifted to the direction of complete splitting of citrate. Prior to the determination of citrate, pyruvate and oxaloacetate can be assayed in the same mixture.
METHODS

Reagents

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.

DETERMINATION

OF CITRATE

371

VI. 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 enzyme was partially purified in a similar way to tliat described by Dagely (3) or Tate and Datta (10) from Aerobacter aerogcn.es.It was stabilized by addition of a combination of bovine serum albumine, sucrose, and magnesium 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 Boebringer. Stability of Reagents: The NADH solution can be stored for 3 weeks at 0C in the dark. The LDH and MDH suspensions are stable for several months when kept at 0. The citrate Iyase loses ca. 10% of its activity within 1 month when stored in the cold. Procedure for Activity Determinations of Citrate Lyase

Wavelength 366 nm; glass cuvet of 1 cm light path; temperature 25C. The experimental measurements were made against air with an Eppendorf photometer; however, other spectrophotometers may be used. The reagents are pipetted into the cuvet in the following order:
Reagent
Final concentration

2.66 0.06 0.20 0.05 0.01 0.01

ml ml ml ml ml ml

buffer (I) NADH solution(II) ZnClt solution (III) citrate solution (V) LDH suspension (VI) MDH suspension (VII)

95 mM
0.2 mM 2.0 mfif 1.7 mM 2 IU LDH/ml 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 for a 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 measured time (min) X mg enzyme taken X mhl extinction of NhDH units = mg

Procedure for Determination

of Citrate

Materials: Citrate can be determined in wine, fruit juices and other samples with low protein content without any preliminary treatment. Samples from animal tissue, serum, and extracts containing protein must be deproteinized prior to the assay. Deproteinization must be performed with reagents which do not form precipitates with zinc ions. Perchloric acid was found to be suitable. Deproteinization: 5 gm of tissue is homogenized immediately after

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removal 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 of 2 N KOH and kept in an ice bath for 15 min. The precipitated KClO, is filtered off, Assay: Wavelength 366 nm, glass cuvet of 1 cm light path, room temperature. Measurements are made against air with an Eppendorf photometer; however, other spectrophotometers may be used. The reagents are pipetted into the cuvet in the following order:
Reagent Final concentration

2.00 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)

67 mM 0.2 mM 0.1 mM

Water is added to obtain a volume of 2.97 ml and El is read; then 0.01 ml LDH suspension (V) and 0.01 ml MDH suspension (VI) are mixed in. Through the action of these enzymes the pyruvate (Py) and oxaloacetate (OA) present in the sample are removed. The end point in optical density E, is reached within 3 min. From EPy+OA = E, - E,, one can calculate the total amount of pyruvate + oxaloacetate present. The concentration of each individual compound can be measured by separate addition of LDH and MDH. Then 0.01 ml citrate lyase suspension (VIII) is mixed in. Citrate is decomposed within 5-10 min, and the E, is read. The difference in optical density 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 sample since

AEVh! ---Z

cdl/

mg substance ml sample = 3.3 X lo3 cm2/mmole

where

E = optical density coefficient of NADH at 366 nm d = light path of cuvet = 1 cm V = assay volume = 3 ml ZJ= sample volume = 0.02-0.9 ml, and M = molecular weight = 192.1.

DETERMINATION

OF CITRATE

3i3

LDH / / Ext. 366nm /CL MDH

0.500 f 0.400

0.300

0.200 0.100

I,,

I,

10 -b t [min]

15

FIG. 1. Determination

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

Figure 1 shows the changes in extinction determination of citrate in wine.

NOTES

that occurred during the

1. Purity

of Citrate Lyase

Our purified preparations of citrate lyase are practically free (<0.05%) of aconitase, alcohol dehydrogenase, and NADH oxidase. When aconitase is present, cis-aconitate and isocitrate are determined together with citrate. Alcohol dehydrogenase interferes with the assay of citrate by regenerating NADH in samples containing alcohol. Oxaloacetate decarboxylase does not interfere with the assay, since a mixture of LDH and MDH is used for oxaloacetate determination. Higher quantities of pyruvate decarboxylase interfere with the assay, but this may be overcome in samples not containing alcohol by the addition of alcohol dehydrogenase. In this case, citrate can be estimated with the alcohol dehydrogenase reaction after being converted to acetaldehydc according to the reaction sequence 3, which rapidly proceeds in the crude bacterial extracts:

374
citrate citrate lyase

MOELLERING

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oxaloacetate

+ acetate

OA decarboxylase Py decarboxylase ADH

+ ) acetaldehyde f COP (3) (4)

pyruvate + COZ acetaldehyde + NADH

+ H+ -

ethanol f NAD+

2. Determination

of Citrate

Under the conditions given earlier, the citrate cleavage reaches completion within 5-10 min. The amount of Zn++ added to the assay mixture must be limited in the presence of phosphate or carbonate in the samples to avoid precipitation. In these cases, the concentration of citrate lyase should be increased by the same factor by which Zn++ is reduced. Quantities of citrate as low as 0.02 pmole (corresponding to 0.004 mg citric acid) may be assayed with satisfying accuracy (+50/O). The sensitivity of the method may be increased further by measuring the consumption of NADH at 340 nm or by fluorometric estimation.
3. Accuracy of the Method

The accuracy of the method is shown in Table 1, which shows the recovery of citrate found on addition of increasing amounts to a deproteinized homogenate of rabbit liver.
TABLE 1 Recoveries of Citrate Added to Rabbit Liver Homogenate
Citrate added EdI Citrate found, WlOk 70 Recov.

1 ml homogenate + 0.03 + 0.06 + 0.09 +0.12 + 0.15

0.040 0.074 0.110 0.134 0.170 0.202

0.0364 f 0.0007 0.0673 0.1010 0.1224 0.1544 0.1844

103 107 96 98.5 99

To improve the accuracy of the method, the citrate content of the homogenate lacking citrate (Table 1) was determined 10 times. The errors lay within limits of -2 and 41.5%. In wine, which has a higher citrate content, the maximal error was &lPr,.
4. Specificity

of Citrate Lyase

The 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 ethanol at concentrations similar to citrate. None of these substances reacted

DETERMINATION

OF CITRATE

315 of the determina-

or exhibited any interference with tion of citrate by this method. 5. Application

speed and accuracy

of the Method materials of animal and plant

The method was applied to various origin. Table 2 gives our results.
Citrate
Substrate

Content

TABLE of Biological

2 Materials

and of Foods
Citric acid content

Human serum Rat liver Rat heart Rabbit liver Wine (Nahe) Cherry juice (Vitaborn) Apricot juice Blueberry juice I Currant juice Orange juice (fresh) Lemon juice (fresh) Frankfurter sausages SUMMARY

0.022 mg/ml 0.024 mg/gm fresh 0.021 mg/gm fresh 0.021 mg/gm fresh 0.200 mg/ml 0.172 mgjml 4.13 mg/ml 4.48 mg/ml 7.00 mg/ml 13.9 mg/ml 65.X mg/ml 1.3 mg/gm wt.

wt. wt. wt.

A method is described for the rapid determination of citrate in biological material which is based on the complete cleavage of the substance by purified preparations of citrate lyaze in the presence of zinc ions. The assay of citrate lyase is also markedly improved by addition of zinc. The method was applied to a number of animal tissues and fruit juices. It is absolutely specific for citrate.
ACKNOWLEDGMENTS The authors are greatly indebted to Dr. H. U. Brrgmeycr cussions and to Dr. G. Holz for the culture of npproprixte aerogenes. REFERENCES 1. STERN, J. in S. P. Colowick u. N. 0. Kaplan &Ict.hods in Enzymology, Vol. 3, p. 426. Academic Press, New York, 1955. 2. SIEBERT, G. in H. U. Bergmeycr Methods of Enzymatic Analysis, p. 318. Icrlag Chcmie Weinheim and Academic Press, Ncs\v l-ark, 1963. 3. DAGLET, S. in H. U. Bergmeyer Methods of l&z?-matic Analysis, p. 313. Vcrlng Chcmie Weinheim and Academic 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). for many helpful disstrains of Aerohncier

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6. DARON,

MOELLERING

AND

GRUBER

7.
8.

9. 10. 11. 12.

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. EISENTHAL, R., S. S. TATE AND S. P. DATTA, 3.FEBS-Meeting, Warsaw 1966, Abstract F169, p. 264. Academic Press, New York, 1966. HARVEY, R. J. AND E. B. COLLINS, J. Biol. Chem. 238, 2648 (1963). TATE, S. S. AND S. P. DATTA, Biochem. J. 91, 18C (1964). TATE, S. S. AND S. DATTA, Biochem. J. 94, 470 (1965). WHEAT, R. W. AND S. J. AJL, J. Biol. Chem. 217, 909 (1955). GRUBER, W. AND H. MOELLERING, Biochem. 2. 346, 85 (1966)