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).




ing phenomenon is most simply visualized by the appearance of a peak upon the injection of pure solvent, in this ease water, after running an unknown mixture or a standard through the column. The ghost peak often has a retention time similar to that of the unknown or standard solute used in the previous injection. The size of the ghost peak decreases with successive injections of solvent. Numerous treatments have been recommended to suppress ghosting and tailing. Among these the addition of formic acid vapor to the carrier gas or to the sample itself appears to be simple and effective,1,3,5 although it does not eliminate ghosting entirely. Method

The following procedure is used for the quantitative determination of acetate in biologic fluids. Propionie acid is used as internal standard. This is the internal standard of choice after it has been determined that the unknown samples do not themselves contain propionate. If propionate is present in the samples to be assayed, some other acid, such as isobutyric acid, can be used as internal standard. Triehloroacetic acid is used to deproteinize the biologic fluid. Triehloroaeetie acid itself produces several major peaks on the chromatogram, but these emerge well before acetic acid and do not interfere with the acetate analysis. In addition, trichloroaeetie acid produces a very small peak at a position on the ehromatogram which coincides exactly with the position of acetic acid. When we first observed this peak we thought that it may be caused by a very small contamination of the reagent grade trichloroaeetie acid used by us with acetic acid. However, the relative size of this unknown peak remained unchanged after the triehloroacetic acid had been recrystallized twice from heptane. The unknown peak is proportional to the concentration of trichloroacetic acid in the solution injected into the column. If this impurity were acetic acid it would represent 0.001% of the triehloroacetie acid in the solution that is injected onto the column. This amount of contamination is so small that it can be neglected when assaying concentrations of acetate greater than 0.7 mM. However, it must be taken into account when acetate concentrations of the order of 0.1 to 0.5 mM are being measured. Since the area' of the impurity peak is constant, the area of the impurity peak observed in blanks is simply substraeted from the area of the peak in the test sample. This impurity is of such a minor nature that we have not attempted to establish conditions under which it could be separated from acetic acid.




The use of perchloric acid to deproteinize the biologic fluid was also investigated. This reagent itself also yields a number of peaks, including a relatively large one that runs in the region of acetate and another large one that runs in the region of propionate. We have avoided the use of perchloric acid to deproteinize our biologic samples, because of the occurrence of the two unknown peaks.

Apparatus A Perkin-Elmer gas chromatography apparatus, model 900, equipped with flame ionization detector is used by us, but many other suitable instruments are commercially available. The glass columns [1.8 m (6 ft) X 2 mm i.d.] are packed with 10% SP-1200 plus 1% H3P04 on Chromosorb W (80-100 mesh).'~ This material is available from Supelco Inc., Bellefonte, Pennsylvania 16823. The columns are plugged at each end with silanized glass wool. The injection port is glass-lined. Nitrogen is used as carrier gas. The columns are conditioned overnight at 200 ° with carrier gas flowing at a rate of 40 ml/minute. Before use the columns are injected several times with 2-3 t~l of water to clear out any extraneous material. The colmnns are operated isothermally at 105°. The injection port and detector are operated at a temperature of 200 °.

Procedure The following procedure is for a sample volume of 0.5 ml. It can easily be adapted to other volumes. I. 0.5 ml of biologic fluid or standard (e.g., 10 mM sodium acetate in 4% serum albuminS. 2. Add 0.1 ml of internal standard (25 mM sodium propionate or sodium isobutyrate). 3. Add 0.1 ml of 20% trichloracetic acid. 4. Mix well for 1-2 minutes. 5. Centrifuge at 2000 rpm for 10 minutes. 6. Inject 0.4 td of supernatant into the injection port of the gas chromatography apparatus. 7. After the last peak has emerged (usually the internal standard), iniect 2 td of water. If ghosting is observed, repeat injection of water. If ghost peaks of acetate or standard are observed, their area is added to those of the original acetate and standard peaks, respectively.



0 150-

[Acetate] (mM)(o) 4 6 I I

2 I


8 I0 I j ¢ ~ 4 0


30 .~.


o I00




= '~ 50 n.I0

0 0


I 2.0

I 3.0

0 4.0

[Acetate] (mM)(e) FIo. 1. Calibration curve for acetate in the range 0.2-10 raM. Various known amounts of acetate were added to samples of a perfusate which had circulated through a liver for 2 hours, and which contained no measurable amounts of acetate. The area of the small peak in the acetate region which occurs in blanks containing no perfusate and no acetate, and which results from a breakdown product of or an impurity in trichloracetatic acid, has been subtracted. It is equivalent to 0.1 mM acetate. Calculations and R e s u l t s A r e a s u n d e r each p e a k are d e t e r m i n e d g r a v i m e t r i e a l l y or b y some o t h e r s u i t a b l e m e t h o d . A r a t i o of u n k n o w n to i n t e r n a l s t a n d a r d is obtained, a n d the c o n c e n t r a t i o n of a c e t a t e in t h e s a m p l e is c a l c u l a t e d from the r a t i o . A c a l i b r a t i o n curve is shown in Fig. I. C h r o m a t o g r a m s of serial s a m p l e s of a r a t liver p e r f u s i o n are shown in Fig. 2. Note T h e p r o c e d u r e calls for t h e i n j e c t i o n of 0.4 ~] of a q u e o u s sample. T h e response of t h e flame i o n i z a t i o n d e t e c t o r to acetic a c i d m a y v a r y with the a m o u n t of w a t e r i n j e c t e d 2 A l t h o u g h this need n o t be of consequence R. B. H. Wills, I. Chromatogr. Sci. 10, 582 (1972).





Fro. 2. Chromatograms showing the appearance of acetate during a rat liver perfusion. Acetate began to appear in response to the addition of 15 mM ethanol 60 minutes after the start of the perfusion. Samples X, Y, and Z were taken 15, 45, and 60 minutes, respectively, after the addition of ethanol. Each acetate peak is indicated by a. Propionate was added as internal standard; each propionate peak is indicated by p. w h e n i n t e r n a l s t a n d a r d s are used with each sample, we prefer to use a c o n s t a n t v o l u m e for a given set of u n k n o w n s , because for a c o n s t a n t v o l u m e of w a t e r i n j e c t e d the peak area is a l i n e a r f u n c t i o n of the acetic acid c o n c e n t r a t i o n .

Determination of acetate by gas-liquid chromatography.

[~8] ACETATE BY GAS--LIQUID CHROMATOGRAPHY 307 concentration of acetate is determined separately on another sample of the same tissue powder. The r...
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