87, 455-459 (1978)
Determination of Acetaldehyde Presence of Formaldehyde’
DORON DAGAN? Department
und Food Science, Massachusetts Cambridge. Mass. 02139
Received August 1, 1977; accepted February 23, 1978 A procedure is described that enables use of the p-phenylphenol color reaction to determine acetaldehyde in the presence of formaldehyde. The sample is first treated with an acidic 2,4-pentanedione reagent. which selectively removes formaldehyde. The method is applicable to biochemical reactions using tissue preparations.
Formaldehyde and acetaldehyde are well-known products of in vivo and in vitro drug metabolism. When both formaldehyde and acetaldehyde are present in the same system, difficulties arise in analysis due to the similarity of their chemical properties and reactivities. For our current studies on carcinogen metabolism, we required a methodology which would allow the analysis of such two-component solutions. The most widely used method for the determination of formaldehyde in biological reactions is the calorimetric procedure described by Nash (1) and modified by Cochin and Axelrod (2). Depending on the particular study involved, however, this method may lack the desired specificity and sensitivity. Recently, Chrastil and Wilson (3) have developed a procedure for determining formaldehyde, which is both sensitive and specific. In addition, its accuracy is not affected by the presence of acetaldehyde in the sample. The widely used calorimetric method for acetaldehyde is the pphenylphenol procedure developed by Barker and Summerson (4) and by Stotz (5). This method, however, cannot be used when formaldehyde is present. Furthermore, with biological samples such as blood, the method * This research was supported by Public Health Service Grant 2-POl-ES00597-07 and Training Grant I-T32-ES07020-02 from the National Institute of Environmental Health Sciences. * Present address: Chemical Industry Institute of Toxicology. Research Triangle Park, N.C. 27709. 3 Author to whom correspondence should be addressed. Recipient of a Research Cancer Development Award from the National Institutes of Health (l-K04-ESOO033-OlAl). 455
0003-2697/78/0872-0455$02.00/O Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any fan reserved.
requires purification by distillation in order to remove interfering substances (5). Distillation is a cumbersome and time-consuming operation to carry out, especially when a large number of separate samples must be analyzed. In the present work, a 2,4-pentanedione reagent similar to the one described by Nash (1) is used to convert formaldehyde selectively to 3,5-diacetyl-1,4-dihydrolutidine within a short time at room temperature. The yellow color of this product does not interfere with subsequent steps of the acetaldehyde determination. Formaldehyde in the sample can be estimated separately by the Chrastil and Wilson procedure (3). Together, these procedures provide a self-consistent analytical method for the calorimetric determination of formaldehyde and acetaldehyde when they are present together in aqueous solution. METHODS Reagents and standards. The 2,4-pentanedione reagent is prepared by mixing 30.8 g of ammonium acetate, 5 ml of concentrated sulfuric acid, and 0.4 ml of 2,4-pentanedione and diluting to 100 ml with distilled water. The p-phenylphenol reagent is prepared according to Stotz (5). Stock acetaldehyde solution was prepared by dissolving 50 ml of freshly distilled acetaldehyde in 500 ml of distilled water. The solution was standardized using a literature procedure (6). Stock formaldehyde solution was prepared by boiling paraformaldehyde in distilled water. After cooling and filtering, the solution was standardized by the sodium sulfite method (7). Working standards containing 50 to 300 nmol/ml formaldehyde and acetaldehyde were prepared by appropriate dilution and mixing of the stock solutions. In addition, all working standards contained 1 mg/ml semicarbazide hydrochloride. Analytical procedure. One milliliter of a solution containing formaldehyde and acetaldehyde is mixed with 0.5 ml of 2,4-pentanedione reagent in a test tube. The mixture is allowed to sit at room temperature for 10 to 20 min. Then 0.05 ml of 5% copper sulfate is added, and the tube is placed in an ice bath. Exactly 10.0 ml of concentrated sulfuric acid is added slowly and with shaking. p-Phenylphenol reagent (0.2 ml) is added just above the surface of the liquid, and the resulting precipitate is vigorously dispersed using a vortex mixer. The mixture is incubated at 30°C for 30 min. The tube is then immersed in boiling water for 1.5 min and cooled. The violet color is read at 560 nm against reaction blank. Metabolic studies. Rat liver microsomal incubation mixtures contained the following components in a final volume of 3.5 ml: 0.1 M phosphate buffer, pH 7.4; 105,OOOg purified microsomes containing 3 mg of protein (8); 3.5 mg of semicarbazide hydrochloride; 70 pmol of N-nitrosomethylethylamine as substrate; and an NADPH-generating system consisting of
0.73 mM monosodium NADP, 7.4 mM monosodium glucose &phosphate, 20 mM MgCl*, and 3 units of glucose 6-phosphate dehydrogenase. Incubation was carried out at 37°C. The reaction was terminated by addition of 1 ml of 15% ZnSO, followed by 1 ml of saturated Ba(OH)*, with cooling in an ice bath. The precipitated protein was centrifuged, and the supernatant was used for the aldehyde determinations. RESULTS
Acetaldehyde solutions that contained no formaldehyde were first treated with 2,4-pentanedione reagent and then analyzed for acetaldehyde as described above. These analyses form the standard curve illustrated in Fig. 1, which was linear up to 300 nmol/ml acetaldehyde. Treatment of sample solutions containing both acetaldehyde and formaldehyde with the 2,4-pentanedione reagent resulted in reaction of up to 370 nmol/ml formaldehyde within 10 min at room temperature. Analysis for acetaldehyde then gave absorbance readings that were essentially superimposable on those obtained with solutions containing no formaldehyde (Fig. 1). The removal of larger amounts of formaldehyde may require somewhat longer reaction times, higher temperatures, or higher concentrations of 2,4-pentanedione. Any formaldehyde not consumed in this step will react with p-phenylphenol to give a blue color (maximum absorbance at 600 nm). This coloration will interfere with quantitative estimation of acetaldehyde at 560 nm.
FIG. 1. Absorbance of various concentrations of acetaldehyde phenol reaction: (0) acetaldehyde only: (0) acetaldehyde plus formaldehyde (k 1 standard deviation). All samples were treated reagent.
in the modified p-phenylan equal concentration of with the 2,4-pentanedione
FIG. 2. Time dependence of formaldehyde and acetaldehyde production during the metabolism of N-nitrosomethylethylamine by a rat liver microsomal preparation: (0) formaldehyde; (0) acetaldehyde.
The quantities and concentrations of reagents described above have been chosen to provide optimum formaldehyde-removal capability together with maximum color development. The presence of semicarbazide hydrochloride serves two functions. First, it minimizes loss of formaldehyde and acetaldehyde from sample solutions. Second, it significantly increases the color intensity in the Chrastil- Wilson assay for formaldehyde (3). It has no effect on the p-phenylphenol color reaction. In the latter procedure, significant losses of volatile acetaldehyde may occur after adding sulfuric acid to the assay mixture. To minimize this, external cooling is recommended. We have applied this methodology for acetaldehyde and formaldehyde analysis in our investigations of nitrosamine metabolism with rat liver microsomal preparations. We found that the ZnSO,/Ba(OH), method for protein precipitation was the only one that was compatible with both our modified p-phenylphenol procedure and the Chrastil- Wilson procedure. Recovery of acetaldehyde added to the microsomal preparations was close to 100%. Thus, in the microsomal system, a distillation step prior to analysis of acetaldehyde appears to be unnecessary. In other biological systems, however, distillation may still be required to remove interfering impurities. Figure 2 illustrates an application of the methods in our studies on the metabolism of N-nitrosomethylethylamine. We were able to monitor the simultaneous formation of both formaldehyde and acetaldehyde from this substrate. The aldehydes are formed via microsomal oxidation at the carbon atoms adjacent to the N-nitroso group followed by cleavage of the unstable cr-hydroxynitrosamines (9). As a note of caution, we found that Tris buffer solutions containing semicarbazide spontaneously develop high concentrations of formal-
dehyde over the course of several weeks. Even freshly prepared Trissemicarbazide solutions developed higher than normal formaldehyde background levels during incubations at 37°C. ACKNOWLEDGMENT We thank Dr. Irene Y. Chau for help in preparing this manuscript.
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