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On the Presence of Acetone in Oxidized

Pyridine Nucleotides’

Alcohol dehydrogenase (ADH) activity in tissue samples has been traditionally assayed by the spectrophotometric or fluorometric meas- urement of the rate of NADH formation (1). Gupta and Robinson (2) have described a new method for assaying ADH activity by measuring the acetaldehyde formed using a system similar to that of Burbridge et al. (3) for detecting acetaldehyde in the blood. This method consists of placing semicarbazide hydrochloride, kept at neutral pH to facilitate collection, in the center well of an Erlenmeyer flask. The flask is then sealed after the material containing the acetaldehyde is placed in the outer well. Because of the low boiling point of acetaldehyde (21”(J), the aldehyde vaporizes at room temperature and is trapped in the semi- carbazide. Subsequently, the acetaldehyde semicarbazone content is determined spectrophotometrically.

More recently the semicarbazide reaction was utilized by us to measure the formation of acetaldehyde from ethanol in the presence of micro- somes (4). The complete system included washed microsomes (3 mg protein), 1.5 mM 3-acetylpyridine NAD (AP-NAD), 50 mM ethanol, and 0.1 M NaPO,, pH 7.4, in a final volume of 3 ml. The mixture was incubated in the outer compartment of a center well flask (25 ml) for 20 min at 37°C. The center well of the flasks contained 0.6 ml 0.015 M semicarbazide in 0.16 M potassium phosphate buffer, pH 7.0. The reaction was terminated by the addition of 0.5 ml 70% TCA to the outer well contents. The sealed flasks were allowed to stand overnight at 23”, after which the center well contents were harvested, diluted to 3 ml with water, and the optical density read at 224 rnp in a Zeiss spectrophotometer against a similar preparation TCA’d at zero time.

In the process of examining this system we observed that AP-NAD resulted in the same significant AODzz4 over a very wide range of pH (5.5-11.4) and ethanol concentration (5-250 mM) . Furthermore, as seen in Table 1, the same AODnsr was obtained when the ethanol or the microsomes or the TCA used to stop the reaction was omitted. Only in the absence of the AP-NAD or semicarbazide was there no AODzz4. Hence, the AOD,,, observed when AP-NAD was added appeared to form

‘This work was supported by a grant from The National Institute of Mental Health (MH 16892).

@ 1972 by Academic Press, Inc.

338 SHORT COMMUNICATIONS

TABLE 1 Effect of Various Assay Conditions on Trapping of a Volatile Substance from

Oxidized 3-Acetylpyridine Nucleotide

Incubation system ACDM

Completea Complete, pH 5.4

pH 11.4 + ethanol, 100 mM + ethanol, 200 mM - ethanol - microsomes - ethanol, microsomes - ethanol, microsomes, TCA - ethanol, microsomes, TCA, AP-NAD - ethanol, microsomes, TCA, semicarbazideb

1.530 1.700 1.600 1.500 1.400 1.550 1.575 1.670 1.514 0.000 0.005

0 See text. * Center well contained 0.6 ml water.

independently of ethanol or TCA by a nonenzymic reaction and appeared directly related to the concentration of cofactor present in the outer well as can be seen in Fig. 1.

The absorption spectrum of the semicarbazone resulting from AP-NAD in the outer well differed from that produced by benzaldehyde, acrolein, or glutaraldehyde but not that of formaldehyde, acetaldehyde, acetone, or proprionaldehyde in that the absorption maxima of these latter com- pounds all were around 224 m,u. No other absorption maxima between 200 rnp and 300 rnp were observed for the unknown product.

The same type of experiment was repeated under conditions commonly employed with oxidized pyridine nucleotides in cholesterol synthesis (5), namely incubation at 37” for 2 hr. We found that when the AP-NAD containing solution was shaken at 37” for 2 hr it was possible to obtain an A0DZZ4 similar to that seen when the flasks were left overnight at 23”. Replacement of the semicarbazide with 2% sodium bisulfite during this incubation at 37” and subsequent application of this solution to a gas-liquid chromatography column (6) revealed a peak which in- creased in height as the AP-NAD mixture was incubated. This peak increase was accompanied by an increase in AOD,,, in the semicarbazide solution (Fig. 2). The peak observed on GLC decreased when sodium carbonate was added to reverse the bisulfite reaction and release the volatile component. It was possible to separate this peak from acetal- dehyde bisulfite, ethanol, and methanol but not from acetone bisulfite. Application of the AP-NAD solution itself to the GLC column produced a peak which corresponded to acetone.

An attempt was made to remove the acetone from the AP-NAD by

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1.8 -

I6- I 6

0.6

339

AP-NAD CONC , mh4

Fro. 1. Relation between AP-NAD concentration and AODx4. Various amounts of AP-NAD were allowed to stand overnight at 23°C as described in the text.

lyophilization or negative pressure. Both procedures proved unsuccess- ful. Only shaking at 37” for 2 hr removed the acetone completely.

Other commonly used pyridine nucleotides were examined for the presence of acetone. As can be seen in Fig. 3, NADP and NAD produced AOD,,, in the absence of microsomes and a peak corresponding to

I I

(c-4 1.00

;:-;::-:-:

I I

0 I 2 HOURS OF INCUBA T/ON

0 I 2 HOURS OF INCUBA T/ON

FIQ. 2. Effect of incubation at 37°C on release of volatile material from AP-NAD. For the spectrophotometric assay, center well contained 0.6 ml 15 mM semi- carbazide in 0.16 M KPO,, pH 7.4. For the GLC assay, semicarbazide was replaced by 6.5 ml 2% NaHSOa. 2 pl samples of bisulfate solution were then applied to the column, as described in the text.

340 SHORT COMMUNICATIONS

2.0

t

NADPH EL I

NADP NAPH

FIG., 3. Presence of acetone in pyridine nucleotides. 1.5 rnJf amounts of various cofactors were allowed to stand overnight as described in text for AODz2,. To determine acetone content in the cofactor solution, 2 41 samples of 15 mM amounts of various cofactor solutions were applied to the GLC column, as described in the text.

acetone by GLC. Again, lyophilization and negative pressure were ineffective in removing the contaminant. Only shaking at 37” for 2 hr removed the acetone completely.

The presence of acetone in oxidized pyridine nucleotides is of significance in studies of alcohol metabolism for a number of reasons. Thus, in the presence of acetone the semicarbazide reaction coupled with spectrophotometry cannot be used to measure the formation of acetalde- hyde from ethanol because acetone semicarbazone gives an absorption spectrum similar to acetaldehyde semicarbazone. Furthermore, the pres- ence of acetone in NADP may help explain the difficulty of demonstrating alcohol dehydrogenase activity (ethanol to acetaldehyde measured by NADPH formation) in crude tissue homogenates with NADP as the cofactor since ADH can oxidize NADPH with acetone (1). Finally, acetone contamination of NAD may help to explain the low activity of ADH (ethanol to acetaldehyde measured by NADH formation) at pH 7.4 and 23” because ADH can oxidize NADH with acetone (1).

REFERENCES

1. BONNICHSEN, R. K., AND BRINK, N. G., in “Methods in Enzymology” (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 1, p. 495. Academic Press, New York, 1955.

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2. GUPTA, N. K., AND ROBINSON, W. G., Biochim. Biophys Acta 118, 431 (1966). 3. BURBRIDGE, T. N., HINE, G. H., AND SCHIEL, A. F., J. Lab. Clin. Med. 35, 983

(1950). 4. ISSELBACHER, K. J., AND CARTER, E. A., Biochem. Biophys. Res. Commun. 39,

530 (1970). 5. BUCHER. N. R., AND MCGARRAHAN, K., J. Biol. Chem. 222, 1 (1956). 6. BONNICHSEN, R., AND LINTURI, M., Acta Chem. Stand. 16, 1289 (1962).

EDWARD A. CARTER

KURT J. ISSELBACHER

Departments of Medicine Harvard Medical School and Massachusetts

General Hospital (Gastrointestinal Unit) Boston, Massachusetts O%i4

Received June 6. 1971

Determination of Carbonate-14C by Liquid Scintillation Counting1

The direct measurement by liquid scintillation counting of radioactive carbonate present in solutions of inorganic salts is made difficult by the insolubility of carbonate salts in scintillation solutions. Procedures used to overcome this difficulty are based principally on the use of gelling or emulsifying agents to keep the insoluble carbonate in suspension (1,2) or preliminary distillation or diffusion into an organic base (3-5) with subsequent transferral to a scintillation vial. Use of gelling or emulsify- ing agents for stabilization of scintillation mixtures or of toluenejcello- solve or dioxane scintillation mixtures in which there may be incipient precipitation is accompanied by progressive decrease in counting rate with time (6). Count-rate is also reported to be influenced unexpectedly by the presence of small amounts of contaminating substances (4,7,8). Preliminary distillation or diffusion into an organic base would appear to be a preferred method of measurement. We have developed such a method, which is simple and of high accuracy and is especially well adapted to measurement of radioactive carbon dioxide formed in metabo- lism, as it either is present as bicarbonate in blood or is collected as respiratory carbon dioxide in a solution of sodium hydroxide.

‘Stipported in part by Research Grant AM-00613 from the U. S. Public Health Servi’ce.

@ 1972 by Academic Press, Inc.