302

GENERAL ANALYTICAL METHODS

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Determination

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of Acetate

By ROBERTW. GVYNN and RICHARDL. VEECH Several enzymatic acetate assays have been previously described. 1-s However, those methods which have been used to determine acetate in tissue extracts have required the distillation or diffusion of the acetate from the extract first before measurement. Besides being time consuming the distillation or diffusion procedures carry with them the very real risk of breakdown of tissue acetyl compounds to acetate during the relatively prolonged, harsh conditions used. Since significant quantities of labile acetyl compounds exist in animal tissues, the breakdown of such compounds is a potential source of error in acetate measurement. In contrast, the following spectrophotometric and fluorimetric procedures for acetate measurement avoid the diffusion step and can be performed directly in the tissue extract.

Assay Methods

Principle.

The method is based upon the enzyme sequence:

A c e t a t e - + A T P 4- + CoA = A S I P ~- + PPi 4- -~- acetyl-CoA + H + Acetyl-CoA + oxalacetate 2- = citrate 3- + CoA -{- H + Malate 2- -{- NAD + = oxalacetate 2- -[- N A D H + H + Net: a c e t a t e - + A T P 4- + malate 2- + N A D + = A M P ~+ PPi 4- + citrate 3- + 3H + + N A D H N A D H production is followed spectrophotometrically or fluorimetrically. The assay depends upon the conversion of acetate to acetyl-CoA and the measurement of the acetyl-CoA by a coupled system of malate dehydrogenase and citrate synthase. The assay is specific since citrate synthase is specific for acetyl-CoA. A potential lack of stoichiometry in R. W. yon Korff, J. Biol. Chem. 210, 539 (1954). I. A. Rose, M. Grunberg-Manago, S. R. Korey, and S. Ochoa, J. Biol. Chem. 211, 737 (1954). 3 I. A. Rose, Vol. 1 [97]. 4 M. Soodak, Vol. 3 [48]. 5 F. Lundquist, U. Fugmann, and H. Rasmussen, Biochem. J. 80, 393 (1961). o H. U. Bergmeyer and It. Moellering, Biochem. Z. 344, 167 (1966). 7 M. Schulman and H. G. Wood, Anal. Biochem. 39, 505 (1971). 8 F. J. Ballard, O. H. Filsell, and I. G. Jarrette, Biochem. J. 126, 193 (1972).

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&cI~.T&TI~. ASSAY

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this system accounts for the lack of stoichiometry in the previously reported use of this system as an acetate assay. 1 This lack of stoichiometry has been discussed a number of times. ~,'~-11 From a practical point of view, however, the inclusion of NADH in the reagent cocktail for the spectrophotometric method produces a stoichiometric assay as judged by the excellent agreement (within 2%) between the assay of standard potassium acetate by the procedure outlined below with both the value expected from the dry weight of the salt and with the value obtained by the acetate assay of Ballard et al. 8

Enzymes and Reagents I. E n z y m e s . Purified beef heart mitochondria acetyl-CoA synthetase (EC 6.2.1.1) is prepared by the method of Webster 12 (specific activity 35 units13/mg of protein at 38°C). Malate dehydrogenase (EC 1.1.1.37) from beef heart (1100 units/mg of protein) and citrate synthase (EC 4.1.3.7) from pig heart (110 units/mg protein) are commercially available. The enzymes used for the assay should have no detectable activity of acetyl-CoA deacylase, NADH oxidase, oxalacetate decarboxylase, carnitine acetyltransferase (EC 2.3.1.7), or ATP citrate lyase (EC 4.1.3.8). 2. R e a g e n t s . Stable for at least a month unless otherwise noted. (a) Stored at 0-4 ° Tris-HC1, 0.2 M, pH 8.0 containing KC1, 0.1 M Magnesium chloride, 0.1 M NADH, 5.0 mM in 0.3% (w/v) KHCO~, stable for 5-7 days Perchloric acid, 1.2 M (b) Stored below --15°C Adenosine 5'-triphosphate, 27 mM, Tris, potassium or sodium salt NAD +, free acid, 24 mM Malate, potassium salt, 0.1 M Coenzyme A, free acid, 1.2 mM containing mercaptoethanol, 3.0 mM, prepare fresh weekly Bovine serum albumin, 100 mg/ml Tissue Preparation. Tissue samples obtained with rapid freezing tech-

W. Buckel and H. Eggerer, Biochem. Z. 343, 29 (1965). loD. J. Pearson, Biochem. J. 95, 23c (1965). 11R. W. Guynn, H. J. Gelberg, and R. L. Veech, J. Biol. Chem. 248, 6957 (1973). 1"~L. T. Webster, Jr., J. Biol. Chem. 9.40, 4158 (1965). J~One unit of enzyme is the amount which will utilize 1 tLmoleof a substrate per minute under optimal conditions.

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niques are used. 14,15 The frozen tissue is powdered under liquid nitrogen, homogenized in 4 volumes of ice-cold perchloric acid (1.2 M) and centrifuged at 35,000 g for 15 minutes at 0% The resulting supernatant is carefully neutralized to pH 6.5 with KOH taking care not to allow the extracts to become basic. After standing at 0 ° for half an hour, the potassium perchlorate precipitate is packed by centrifugation and the resulting supernatant is used for the assay. I n s t r u m e n t s . The spectrophotometric assay is designed for either a dual or single beam instrument holding cuvettes of 1 cm light path. The fluorimetric procedure is designed for an instrument capable of handling 1.0 ml volumes. For the fluorimetric assay quinine sulfate in sulfuric acid is used as the reference standard16; however, for each assay a standard curve of potassium acetate is run simultaneously. Spectrophotometric Procedure Perchloric acid extracts of tissue containing 0.008-0.1 gmole acetate per milliliter can be satisfactorily measured with the spectrophotometric procedure. This range corresponds to tissue concentrations of approximately 0.04-0.5 gmole/g wet weight tissue. Considerably higher concentrations can be measured although the time required for completion will be prolonged making it more convenient in such a situation to dilute the extract. A reagent cocktail is prepared containing: 90 mM Tris pH 8.0, 2.7 mM ATP, 0.075 mM NADH, 0.6 mM NAD ÷, 0.12 mM CoA, 0.6 mM potassium malate, 4.5 mM MgC12, 0.3 mM mercaptoethanol, 45 mM KC1, 50 mg/ml dialyzed bovine serum albumin, 12 nnits/ml malate dehydrogenase, and 0.5 unit/ml citrate synthase. In the complete reagent cocktail acetate contamination from the reagents is expected to be less than 3 nmoles/ml. Two parts cocktail are combined with one part tissue extract in a cuvette of 1 cm light path. Usually a final volume of 2.1 ml has been found convenient; however, the lower limit for the final volume is restricted only by the size of the euvette (of 1 cm light path) which can be used with a particular spectrophotometer. The blank cuvette contains cocktail plus water. After an initial reading at 340 nm, 0.075 unit of acetyl-CoA synthase is added per milliliter of the complete reaction mix14A. Wollenberger, O. Ristau, and G. Schoffa, Pfliiegers Arch. Gesamte Physiol. Menschen Tiere 270, 399 (1960). is R. L. Veech, R. L. Harris, D. Veloso, and E. H. Veech, J. Neurochem. 20, 183 (1973). ~60. H. Lowry and J. V. Passonneau, "A Flexible System of Enzymatic Analysis," pp. 7-8. Academic Press, New York, 1972.

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ACETATE ASSAY

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ture, and the reaction is followed to completion (15-20 minutes typically). The reaction is stoichiometric; therefore, the concentration acetate in the tissue extract can be calculated directly from the AOD and the extinction coefficient of NADH (~ = 6.22 X 106 cm-~/mole).17 Concentration of acetate in tissue extract (in gmoles/ml) (~OD) 6.22 (fraction of final volume due to extract) When measuring acetate in tissue extracts the A0D is relatively low, often in the range of 0.030-0.120. However, in spite of the relatively low ±OD, high precision and accuracy are possible. On triplicate samplings of the same frozen tissue powder the standard error of the mean of the three determinations with the spectrophotometrie assay is less than 1%. Assays of standard acetate solutions show similar precision. The precision of the method seems not to be as much a function of the assay as it is a function of the care with which the tissue is prepared. Using standard acetate solutions the l},nearity of the assay has been confirmed between optical density changes of 0.030 and 0.400. The accuracy of the method is supported by the finding that the values obtained for the standard acetate solutions agree within 2% of the values expected from the dry weight of the standard potassium acetate and the values obtained by the method of Ballard et al. s Fluorimetric Procedure For low concentrations of acetate in a tissue extract (0.003-0.012 gmole/ml, corresponding to tissue concentrations or approximately 0.015-0.05 ~mole/g wet weight) a fluorimetric procedure is preferred. The reagent cocktail is modified to contain 66 mM Tris-HC1 pI-I 8.0, 33 mM KC1, 0.050 mM potassium malate, 0.025 mM NAD +, 3.0 mM MgCI~, 0.2 mM mercaptoethanol, 0.04 mM CoA, 1.0 mM ATP, 0.1 mg/ml bovine serum albumin, 3.5 units/ml malate dehydrogenase and 0.15 unit/ml citrate synthase. One milliliter cocktail is combined with 100 ~1 extract (equivalent to approximately 15 mg wet weight tissue). After a base line reading 0.03 unit of acetyl-CoA synthetase is added and the reaction is followed to completion (40-60 minutes). A standard curve of potassium acetate is run simultaneously and used to calculate the acetate concentrations in the extracts. Often brain extracts assayed fluorimetrically can be used directly after neutralization. However, treatment of the neutralized extracts with Florisil 1~ (50 mg/ml extract) followed by centrifuga1~B. L. Horecker and A. Kornberg, J. Biol. Chem. 175, 385 (1948). ~s100-200 mesh, Fisher Scientific Co., Fairlawn, N.J.

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tion at 0 ° and 15,000 g for 20 minutes reduces the background fluorescence of an extract without a detectable loss of acetate. If tissues containing a large amount of flavins are to be analyzed fluorimetrically, the preliminary treatment of the extract will be necessary. The fluorimetric procedure is usually less precise than the spectrophotometric assay. The standard error of the mean of triplicate tissue samples is 3-5%. The higher standard error of the mean in the fluorimetric procedure reflects the relatively greater significance of the acetate contaminations of the reagent cocktail at these very low concentrations of acetate.

Variations of the Procedures The actual concentrations of the activating ions Tris and K ÷ do not seem to be critical although the total is best kept above 80 mM. 19,2° On the other hand, Na ÷ concentrations above 5 m M should be avoided even in the presence of optimal K ÷ or Tris. High Na ÷ concentrations will slow the assay making the end point less distinct and producing the potential hazard of an underestimation of the amount of acetate present. Such a problem can be readily detected by using internal standards. The assay procedures as described have been developed for use in such tissues as liver and brain; however, the procedures should have general applicability to the perchloric acid extract of any tissue. Difficulty might be anticipated, however, with tissues with a high sodium content (e.g., blood). If Na ÷ inhibition is considered important in such cases, the concentration of acetyl-CoA synthetase can be increased as needed. The extracts of most solid tissues, however, contain sufficiently low concentrations of Na t that even Na~ATP.2½H20 can be used in the reagent cocktail without serious interference. The concentrations of malate and NAD ÷ should not be significantly increased in the spectrophotometric assay since the stoichiometry of the assay m a y be compromised. The usual concentrations of malate and NAD ÷ in tissue extracts do not interfere.

Recoveries Known amounts of potassium acetate equivalent to tissue concentrations are added to frozen tissue powder. The tissue is then extracted with perchloric acid and assayed by the standard procedure. The endogenous 1~R. W. yon Korff, J. Biol. Chem. 203, 265 (1953). 2oF. Campagnari and L. T. Webster, Jr., J. Biol. Chem. 238, 1628 (1963).

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ACETATE BY GAS--LIQUID CHROMATOGRAPHY

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concentration of acetate is determined separately on another sample of the same tissue powder. The recovery of the small quantities of acetate added to the frozen tissue is 95-98%. If acetyl-CoA, acetylcarnitine, or N-acetyl-aspartate is added to the frozen tissue in amounts equal to those normally found in tissue, they do not interfere with the determination of the acetate. There is no evidence for breakdown of these compounds to acetate during the extraction procedure or the assay. Discussion

The relatively low Km of acetyl-CoA synthetase for acetate makes the direct measurement of acetate in tissue extracts practical (K,~ -- 0.8 mM).l~ Normal acetate values vary considerably among tissues and dietary states; for example, the following values have been found in rat liver: starved 48 hours, 75-130 nmoles/g wet weight; fed ad libitum, 125-200 nmoles/g wet weight; meal-fed (3 hours daily), 175-275 nmoles/g wet weight; starved and then refed a no-fa~ high sucrose diet, 20-30 nmoles/g wet weight. In rat brain lower values have been found and there is less obvious variation with diet: starved 48 hours or fed ad libitum, 20-30 nmoles/g wet weight. 21 ~' For further details of normal tissue values, recoveries, and the procedure see R. W. Guynn and R. L, Veech Anal. Biochem. (1974) (in press).

[38] D e t e r m i n a t i o n o f A c e t a t e b y G a s - L i q u i d C h r o m a t o g r a p h y

By M. WADKE and J. M. LOWENSTEI~ Analysis of aqueous solutions of short-chain free fatty acids (C2 to C6) by gas-liquid chromatography has been reported by numerous workers. 1-6 The quantitative determination of acetate in biologic fluids is subject to a number of errors. Potentially serious sources of error arise from the tailing of peaks and the formation of ghost peaks. Tailing of peaks is easily recognized and has been discussed elsewhere. 7,~ The ghost1 R. G. Ackman, J. Chromatogr. Sci. 10, 560 (1972). 2 D. M. 0ttenstein and D. A. Bartley, J. Chromatogr. Sci. 9, 673 (1971). s D. A. M. Geddes and M. I~. Gilmour, J. Chromatogr. Sci. 87 394 (1970). 4 V. Mahadevan and L. Stenroos, Anal. Chem. 39, 1652 (1967). R. G. Ackman and J. C. Sipos, J. Chromatogr. 137 337 (1964). 6 R. Turner and M. N. Gilmour, Anal. Biochem. 13, 552 (1965). 70. E. Schupp, in "Gas Chromatography" Tech. Org. Chem. (E. S, Perry and A. Weissberger, eds.), p. 13. Wiley (Interseience), New York (1968). 8 D. M. Ottenstein, Advan. Chromatogr. 3, 141 (1968).

Enzymatic determination of acetate.

302 GENERAL ANALYTICAL METHODS [37] E n z y m a t i c Determination [37] of Acetate By ROBERTW. GVYNN and RICHARDL. VEECH Several enzymatic acet...
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