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Home , Acetaldehyde, Ethanol

Food 201 (2016) 270–274

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Food Chemistry

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Analytical Methods Detecting and by simple and ultrasensitive fluorimetric methods in compound foods ⇑ M. Zachut, F. Shapiro, N. Silanikove

Biology of Lactation Laboratory, Department of Ruminant , Institute of Animal Sciences, Agricultural Research Organization, Volcani Center, Bet Dagan 50250, Israel article info abstract

Article history: There is a need for simple, accurate, and rapid analysis of ethanol (Eth) and acetaldehyde (AA) in a wide Received 15 July 2015 variety of beverages and foods. A novel enzymatic assay coupled to formation of fluorescent chro- Received in revised form 20 October 2015 mophore is presented. Eth detection was further improved by adding semicarbazide to the reaction mix- Accepted 19 January 2016 ture, which interacts with AA and prevents its inhibitory effect on Eth oxidation. The limits of detection of Available online 21 January 2016 Eth (0.5 mg/L) and AA (0.9 mg/L) are comparable with the performance of modern gas chromatography techniques. The repeatability of Eth and AA detection in various foods (9% on average) was lower than Keywords: that with commercial kits (23%). The high sensitivity of the developed method enables detection of AA Ethanol in common foods [e.g., bio-yogurt (12.2 mg/L), and the existence of endogenous Eth (1.8 mg/L) and AA Acetaldehyde Fluorimetric assay (2.0 mg/L) in -free non-fermented bovine milk], which could not measured so far by enzymatic methods. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction instruments (HPLC). GC/FID has been used for the determination of Eth and AA and in and beverages without sample Ethanol (Eth) and acetaldehyde (AA) are metabolites, which are preparation; however, it requires relatively high sample volumes produced during processes and are commonly pre- ( 450 ll) and dedicated equipment (Homann et al., 1997; sent in fermented beverages and foods. They may therefore be pre- Linderborg, Salaspuro, & Väkeväinen, 2011). A particular problem sent in humans’ biological fluids. In mammals, AA is the main with the GC/FID method is formation of artificial AA due to Eth oxi- product of Eth oxidation in the (Hipólito, Sánchez, Polache, dations, which requires particular procedures to account for the & Granero, 2007). Accurate measurements of Eth and AA are there- problem (Fukunaga, Silanaukee, & Eriksson, 1993). Consequently, fore required in a variety of matrices, such as alcoholic beverages, the availability of an analytical method that is simple, rapid, foodstuffs, , and pharmaceuticals. Moreover, measure- cost-effective and accurate for the determination of Eth and AA is ment of Eth and AA in the blood plasma and other biological fluids desirable. AA is a toxic substance, a class 1 and muta- is of particular importance for the diagnosis and treatment of genic at concentrations of 50–100 lM (2.2–4.4 mg/L) (Seitz & alcohol-use disorders, as biomarkers for several diseases, in Stickel, 2007). Recently, mutagenic levels of AA have been reported acute intoxications, and in forensic settings (Schlatter, Chiadmi, in various foods (Lachenmeier & Sohnius, 2008; Uebelacker & Gandon, & Chariot, 2014). Lachenmeier, 2011). Although the detection limit of modern GC Many methods, such as gas-diffusion flow-injection analysis methods is sufficient to detect mutagenic levels of AA in foods (FIA), electroanalysis, FIA-electroanalytical detection, infrared (IR) (Lachenmeier & Sohnius, 2008; Pontes et al., 2009), this method , direct injection gas chromatography (GC)/flame ion- is quite cumbersome for routine analyses. Conversely, current ization detection (FID), headspace injection GC/FID, high- enzymatic analyses, with detection limits above 10 mg/L (Beutler, performance chromatography (HPLC)/Fourier transform 1988), are not suitable for detecting mutagenic levels of AA. Thus, (FT) and others have been developed for analyses of Eth and AA the need for a fast and versatile method to determine low levels of (Jain & Cravey, 1972a, 1972b; Ramdzan, Mornane, McCullough, AA in various beverages and foods is of particular importance. Mazurek, & Kolev, 2013; Schlatter et al., 2014). However, some of Enzymatic methods that utilize alcohol (ADH) these methods are not sufficiently accurate (e.g., older versions of and AA dehydrogenase (AADH) are well-known and frequently GC; Schlatter et al., 2014), some require complex and expensive used to analyze Eth and AA in biological specimens (Beutler, 1988; Redetzki & Dees, 1976). ADH oxidizes Eth to AA and AADH

⇑ Corresponding author. oxidizes AA to acetic . Both use nicotinamide + E-mail address: [email protected] (N. Silanikove). dinucleotide (NAD ) as coenzyme, which is reduced during the http://dx.doi.org/10.1016/j.foodchem.2016.01.079 0308-8146/Ó 2016 Elsevier Ltd. All rights reserved. M. Zachut et al. / Food Chemistry 201 (2016) 270–274 271 reaction to form NADH. NADH formation is stoichiometrically 2.3. Reaction mixtures linked to the oxidation of Eth and AA. Thus, the concentration of NADH in specimens can be used to monitor the concentrations of All reagent were prepared fresh once a week with metabolites formed by NAD+ dependent spec- double-distilled . For Eth and AA determinations according to trophotometrically or fluorometrically (Beutler, 1988; Redetzki & Option 1 (Fig. 1), the reaction mixture was composed of 1 mM Dees, 1976; Shapiro, Shamay, & Silanikove, 2002). However, mea- NAD+,48lM resazurin, 1U/mL, diaphorase, 100 mM KCl, 0.0004% suring NADH in foods and other matrices is frequently difficult (w/v) Triton X-100, and 50 lL ADH (10 kU/mL) and 10 lL AADH and problematic because: (i) these substances contain fat droplets (75 U/mL) dissolved in 50 mM potassium phosphate buffer, pH 7.6. of varying sizes that scatter light in an unpredictable way; (ii) as a For the separate determination of AA, addition of ADH was omitted . result of their opaque and colloidal properties, they scatter and InOption2 (Fig.1), thestocksolutionforAAwaspreparedwithout absorb light; (iii) they frequently contain intense colorants that ADH and contained 75 mM semicarbazide and 130 mM . interfere with the monochromatic absorbance. NADH can be deter- mined by fluorometric means, which are free of these limitations. 2.4. Standard curves However, because additional indigenous biological substances emit light in the same range, fluorometric determination of NADH Stock solutions were prepared by dissolving 100 mg/mL of Eth is associated with considerable background noise, which reduces or AA in double-distilled water. Standards were prepared by seri- the sensitivity of the method (Shapiro & Silanikove, 2010, 2011; ally diluting the stock solutions of the test substances in distilled Shapiro et al., 2002). water to concentrations of 1, 2.5, 5, 10, 25, 50, 100, 250, A general for measuring metabolites that are involved 500 and 1000 mg/L. in reactions of NAD+-coupled dehydrogenases is to combine the reaction to another set of coupling reactions: diaphorase (EC 2.5. Reaction procedures 1.6.99.1) oxidizes NADH to NAD+, and this reaction can be coupled to the conversion of non-fluorometric resazurin to the highly fluo- All procedures were carried out in the wells of a 96-well micro- rochromophoric substance resorufin (Shapiro & Silanikove, 2010, plate suitable for fluorometric reading. Reaction mixture (100 lL) 2011; Shapiro et al., 2002). To date, this methodology has been and test solution (10 lL) (standard or test samples) were incubated found useful for accurate determination of D- and L-lactate, , together in the wells for 30 min at room temperature. The plates galactose citrate, malate pyruvate and oxaloacetate in milk, were read in a fluorometer (ELx800, BioTek Instruments, Winooski, yogurts and colored , such as red and , without VT, USA) at excitation and emission wavelengths of 540 and the need for pretreatments (Shapiro & Silanikove, 2010, 2011). 590 nm, respectively. Oxidation of Eth is particularly sensitive to inhibition by its pro- duct, AA (Kristoffersen, Skuterud, Larssen, Skurtveit, & Smith- Kielland, 2005; Kristoffersen & Smith-Kielland, 2005). A potential 2.6. Biological and food samples solution to this problem is to force the reaction toward completion, thereby overcoming product inhibition. When semicarbazide was Milk was sampled from the commingled milk of six cows with added to a reaction solution containing AADH and NAD+, it reacted bacteria-free udders. Bacteria-free samples were defined as the with AA to form semicarbazone, which does not inhibit the reaction rate (Kristoffersen & Smith-Kielland, 2005; Kristoffersen et al., 2005). In that modification, NADH was determined spectrophoto- metrically, suggesting that the sensitivity and range of biological sources without sample preparation might be improved by apply- ing the fluorometric determination of resorufin, as already noted. Hence, the objective of this study was to apply the above- described modifications to improve the detection of Eth and AA in compound beverages and foods.

2. Materials and methods

2.1. Chemicals

The following chemicals were obtained from Sigma (Rehovot, Israel): ADH (EC 1.1.1.1), AADH (EC 1.2.1.5), diaphorase from Pseu- domonas fluorescens (100 U/L), AA, Eth (AA and Eth standards are stored in ampoules), glycine, KCl, b-NAD+ , resazurin, Trizma base, and Triton X-100. In addition, two commercial assay kits were purchased: ethanol assay kit (MAK076 Sigma) and acetaldehyde assay kit (Megazyme International, Bray, Ireland). The concentrations of Eth and AA in the samples tested by the commercial kits were determined according to the manufacturer’s instructions after appropriate dilution, as described below. Fig. 1. Schematic representation of assay principles for the determination of ethanol (A) and acetaldehyde (B). Footnote: in the conventional mode (Option 1), 2.2. Assay principle the reactions in (A) and (B) are carried out in sequence without the addition of semicarbazide (yielding the concentration of ethanol + acetaldehyde), and the reaction in panel B (yielding the concentration of acetaldehyde). The concentration The enzymatic reactions that served as the basis for the analysis of ethanol is calculated as the difference between (A + B) – B. In the modified mode of Eth and AA and a schematic representation of the reaction (Option 2), ethanol and acetaldehyde are determined separately as described in cycling are shown in Fig. 1. panels (A) and (B). 272 M. Zachut et al. / Food Chemistry 201 (2016) 270–274 absence of bacterial identification over three samplings taken once was determined using an added external standard. The samples every 3–4 weeks. The sampling procedure and bacterial identifica- were spiked at two levels (1 and 10 lM), each in triplicate with tion were carried out according to internationally recognized stan- known quantities of the test compound, and the percentage recov- dards (Leitner, Krifucks, Merin, Lavi, & Silanikove, 2006). The milk ery was calculated. The percentage of recovery rate for the tested sampled for chemical analysis was stored on ice, skimmed compounds was established from the experimental response val- (Silanikove & Shapiro, 2007) and analyzed as described below. ues [(blank + standard) À blank] obtained according to the calibra- Blood was taken from these cows into heparinized tubes and the tion curves and the real concentration of the standard added. Each plasma was separated by centrifugation. of the foods was analyzed in triplicate, each time on 3 separate In addition, the concentrations of Eth and AA were determined in days. Within-day and between-day repeatability was defined as the following commercial sources: kefir (locally produced), bio- the SD of the respective measurements and presented as SD as per- yogurt (fermented milk with addition of probiotic; locally produced, centage of the mean (RSD). Statistical differences between values international brand), beer (lager, locally produced, international of the same sample measured by different methods were obtained brand), and (alcoholic , produced in ). In these by paired t-test. products, the concentrations of Eth and AA were also determined by commercial kits. Additional commercial sources that were ana- lyzed for Eth and AA concentration were: wine (Merlot, local brand), 3. Results and discussion (produced in Russia), synthesized (locally produced), vinegar from apple fermentation (locally produced), balsamic 3.1. The standard curves vinegar (produced in Italy), lemon juice (locally produced), cola soft drink (locally produced, international brand), and an energy drink Determination of Eth using semicarbazide in a separate reaction (locally produced, international brand). (Option 2) proved advantageous compared to the combined reac- 2 For the kefir and bio-yogurt, fluid was extracted by centrifuga- tion (Option 1) in terms of higher R of the calibration curve, lower tion and used for analysis. For the other sources, the only prepara- LOD and greater linear range of the reactions (Represented by tion was dilution to fit the linear portion of the standard curves. upper limit of detection, ULD, in Table 1), which was at least twice As a precautionary measure against the possibility of false as large with Option 2 (Table 1). In general, the improved LOD, LOQ results, we also performed a recovery analysis of spiked Eth and and range of linearity obtained using Option 2 was consistent with AA by applying the levels found in the milk and carrying out the similar improvement attained by converting the colorimetric standard curve analysis (Option 2 in the case of Eth) using milk detection of NADH to fluorometric methods (Shapiro & as the medium. Silanikove, 2010, 2011; Silanikove & Shapiro, 2012). The LODs of Eth and AA were compatible with those obtained by modern GC methods (Lachenmeier & Sohnius, 2008; Pontes et al., 2009) and 2.7. Calculation, validation parameters and statistical analysis were markedly better than those obtained with currently available enzymatic methods (Beutler, 1988; Redetzki & Dees, 1976). Thus, The concentrations of the analyzed metabolites were derived our method enables the detection of Eth at the level required for from linear regression analysis of the calibration curves. For the forensic settings and the reliable detection of mutagenic levels of determination of linearity, regression lines were calculated as AA in foods (Tables 2 and 3). y = a + bx, where x was concentration, and y the response. Ten con- centration points in triplicate were used to prepare the calibration curves. For each compound, the coefficient of determination (R2) 3.2. Comparison of developed method and commercial kits was calculated and the repeatability was assessed based on the rel- ative standard deviation (RSD) values for the corresponding We compared Eth and AA concentrations in commercial bever- response factors. A blank was run in five replicates and its values ages and foods as analyzed by commercial kits for Eth and AA, by were subtracted from the readings. The limit of detection (LOD) Options 1 and 2 for Eth, and by the developed fluorescent method for each metabolite was determined by calculating the y value for AA (Table 2). The commercial kit for Eth determination worked (concentration) derived from blank + 3 SD of the blank. The limit on the same principle as our method using Option 1, although the of quantification (LOQ) was derived from the blank + 10 SD of the manufacturer did not disclose the reagents used, including those blank. In the case of milk, we used the intercept to calculate LOD for the fluorescent coupling reaction. The commercial kit used for and LOQ. Repeatability of the assay method was analyzed by calcu- AA was a colorimetric version of the method developed here. lating the RSD values of three replications of the standard curve Within each compared source, there was no significant difference analysis. The upper limit of linearity was determined when the between either Eth or AA concentrations determined by the differ- expected response (based on the linear regression of lower concen- ent methods. The concentration of AA in the bio-yogurt was three tration points) was smaller than expected by 3 RSD. Day-to-day times higher than the concentration of 100 lM AA (4.4 mg/L) repeatability was estimated by calculating the RSD derived from which is considered to be mutagenic (Lachenmeier & Sohnius, analyses of the standard curve over 3 consecutive days. Recovery 2008; Seitz & Stickel, 2007).

Table 1 Linearity (R2), limit of detection (LOD), limit of quantification (LOQ), upper limit of detection (ULD), accuracy (RSD of the estimate) and repeatability (day-to-daya of RSD) of ethanol and acetaldehyde.

Substance R2 LOD (mg/L) LOQ (mg/L) ULD (mg/L) RSDb of the estimate (%) Day-to-day RSD (%) Ethanol, Option 1 0.991 0.6 2.0 50 5 7 Ethanol, Option 2 0.999 0.5 1.6 100 3 4 Ethanol, Option 2, in milk 0.998 0.5 1.7 100 4 4 Acetaldehyde 0.998 0.9 3.1 100 4 5 Acetaldehyde in milk 0.997 1.0 3.2 100 5 6

a Same measurements over 3 days. b RSD – relative standard deviation. M. Zachut et al. / Food Chemistry 201 (2016) 270–274 273

Table 2 Comparison of the concentrations (lg/ml) of ethanol and acetaldehyde in four commercial food and beverage sources as determined by commercial kits and the developed test methods.

Type of analysis Ethanol by kit Ethanol Option 1 Ethanol Option 2 Acetaldehyde by kit Acetaldehyde Source of sample Beer (lager, international brand) Concentration (mg/L) 41120 (4.1%, w/v) 41750 (4.2%, w/v) 41450 (4.1%, w/v) 0.1 0.1 RSD (%) 21.2 41.4 5.7 35.2 12.5 Source of sample Cognac (from France) Concentration (mg/L) 456750 (45.7%, w/v) 450315 (45.0%, w/v) 452980 (45.3%, w/v) 0.7 0.7 RSD (%) 28.2 38.2 9.1 18.1 5.9 Source of sample Kefir (fermented milk, local brand) Concentration (mg/L) 3455 3540 3340 1.4 1.4 RSD (%) 21.1 17.0 9.0 10.3 23.1 Source of sample Bio-yogurt (yogurt with probiotic culture, international brand) Concentration (mg/L) 25.2 22.6 27.5 13.8 12.2 RSD (%) 25.1 17.8 12.2 28.6 9.0 Average RSD (%) 23.9 28.6 9.0 23.0 12.6

Table 3 l Concentrations of ethanol and acetaldehyde ( g/ml) in cow blood plasma, cow fresh levels in alcoholic drinks were consistent with expected levels milk and a variety of beverages and foods. and the values indicated by the producers on the labels. No signif- Source of sample Ethanol RSD Acetaldehyde RSD icant levels of Eth or AA were found in the lemon juice, cola soft (mg/L) (%) (mg/L) (%) drink or energy drink, all non-fermented products. In contrast, Cow plasma Not – Not detected – except in the tested varieties of beer and kefir (Table 2), all tested detected fermented products—bio-yogurt (Table 2), wine, vodka and vine- Cow milk, raw, fresh 1.8 8.0 2.0 7.0 gars (Table 3)—contained mutagenic levels of AA. The millimolar Wine, Merlot, local brand 136020 1.3 11 (250 lM) 6.6 (13.6%, levels of AA in apple vinegar and balsamic vinegar, which are w/v) world-famous for their , were 14- and 34-fold higher than Vodka, Russian brand 665040 1.1 5.5 (125 lM) 12.5 mutagenic levels (Seitz & Stickel, 2007) of this compound (Table 3). (66.5%, In a close note, we would like to remark that further research is w/v) Vinegar, synthesized, local 103.8 2.8 8.1 (183 lM) 14.3 required to correlate between in vitro finding on mutagenic levels brand of AA in foods and carcinogenicity. However, formation of DNA Vinegar, fermented from apple 179.2 2.2 60.0 8.0 adducts from AA (Brooks & Theruvathu, 2005), the prevalence of juice, local brand DNA adducts in the oral cavity, in association with alcohol drinking Vinegar, fermented, balsamic- 69.3 3.1 166.0 2.0 (Balbo et al., 2012) and alcohol associated increases risk of cancer type, Italy Lemon juice, local brand (no 0. 6 25.5 1.8 10.9 of oral cavity and pharynx, esophagus (Bagnardi et al., 2015; additions) Lachenmeier & Monakhova, 2011) and high incidence of esopha- Cola soft drink 7.3 4.7 0.9 10.3 geal cancer in African population consuming fermented milk with Energy drink, international 1.8 15.7 1.7 3.9 high content of AA (Nieminen et al., 2013) strongly suggest that AA brand secondly formed from Eth and high content of AA in food should be considered as risk factor for cancer development in upper parts of The concentrations of Eth in beer and cognac were consistent the gut. Our restricted survey is consistent with a larger one that with the expected levels (Lachenmeier & Sohnius, 2008) and were showed that many common drinks and foods may contain muta- within ±10% of those declared by the producer. However, the genic levels of AA (Lachenmeier & Sohnius, 2008; Uebelacker & pooled RSD (within and between days) of Eth ranged between 4% Lachenmeier, 2011). As applied by Uebelacker and Lachenmeier and 12%, 9% on average, and was considerably lower than that (2011), a digestion step with simulated gastric fluid may be obtained with Option 1 (17–41%, average 18%) or the commercial required to account for AA bound to or other kit (21–25%, average 24%). Similarly, the RSD of AA concentration in food samples. In conclusion, research of the type described in determined by the fluorescent method ranged from 6 to 23%, 9% Lachenmeier and Sohnius (2008) and Uebelacker and on average, which was much lower than the values obtained Lachenmeier (2011) papers may lead to improved food security with the commercial kit (10–35% and 23% on average). It could by convincing regulatory bodies to adjust regulatory roles to find- be concluded that using Option 2 for the Eth determination and ings; for instance, by preventing the addition of pure forms of AA to the developed method for AA offers a considerable improvement foods. The method developed here provides a simple, accurate and in repeatability over the methods to which they were practical means of gaining broad information on AA content in compared, and thus increased assurance that the observed values foods. reflect the actual metabolite concentrations in the samples. The recovery of Eth and AA in the samples ranged between 95% and 106%, which is consistent with previous performance when 3.4. Concentrations of Eth and AA in non-fermented bovine milk similar modifications have been made for the determination of var- ious metabolites (Shapiro & Silanikove, 2010, 2011). No detectable levels of Eth or AA were found in the cows’ blood plasma (Table 3). However, the fresh milk, which was sampled 3.3. Mutagenic levels of AA in some common alcoholic drinks, foods from cows that were free of bacterial infections, contained low and food supplements levels of Eth and AA. The level of Eth was higher than the LOD (P < 0.07) and about equal to the LOQ (P < 0.01) (Chandran & Concentrations of Eth by Option 2 and AA in a range of bever- Singh, 2007). Recovery levels of Eth and AA relative to the level ages and foods are presented in Table 3. As noted in Table 2, Eth found in milk were 98 ± 3% and 91 ± 6%, respectively. LOD and 274 M. Zachut et al. / Food Chemistry 201 (2016) 270–274

LOQ of standard curves made in milk did not differ significantly De Leonardis, D., Lopez, F., Nag, A., & Macciola, V. (2013). Occurrence and from those made in water (Table 1). persistence of in unfermented and fermented milks. European Food Research and Technology, 236(4), 691–697. To the best of our knowledge, this is the first time that Eth has Fukunaga, T., Silanaukee, P., & Eriksson, C. J. P. (1993). Problems involved in the been detected in bacteria-free and non-fermented mammal’s milk. determination of endogenous acetaldehyde in human blood. Alcohol and This might be related to the significant improvement in its detec- , 28(5), 535–541. Hipólito, L., Sánchez, M. J., Polache, A., & Granero, L. (2007). Brain of tion levels. The AA levels were higher than the LOD and were about ethanol and alcoholism: An update. Current Drug Metabolism, 8(7), 716–727. two-thirds of the LOQ and therefore, this result most likely repre- Homann, N., Jousimies-Somer, H., Jokelainen, K., Heine, R., & Salaspuro, M. (1997). sents the actual content of AA in the milk. Furthermore, AA has High acetaldehyde levels in saliva after ethanol consumption: Methodological aspects and pathogenetic implications. , 18(9), 1739–1743. been recently detected in unfermented raw milk of cow, buffalo, Jain, N. C., & Cravey, R. H. (1972a). Analysis of alcohol. I. A review of chemical and goat and sheep (De Leonardis, Lopez, Nag, & Macciola, 2013), infrared methods. Journal of Chromatographic , 10(5), 257–262. although we believe that the reported levels (20–65 mg/L) in that Jain, N. C., & Cravey, R. H. (1972b). Analysis of alcohol. II. A review of gas chromatographic methods. Journal of Chromatographic Science, 10(5), 263–267. case were about 10-fold higher than the actual ones. Kristoffersen, L., Skuterud, B., Larssen, B. R., Skurtveit, S., & Smith-Kielland, A. The content of AA in milk might have been related to the trans- (2005). Fast quantification of ethanol in whole blood specimens by the fer of AA from the air to the bloodstream upon inhalation, further enzymatic method. Optimization by experimental passing into the milk (De Leonardis et al., 2013). Additional possi- design. Journal of Analytical Toxicology, 29(1), 66–70. Kristoffersen, L., & Smith-Kielland, A. (2005). An automated alcohol dehydrogenase ble sources for AA in the milk are via fodder digestion and absorp- method for ethanol quantification in and whole blood. Journal of Analytical tion (De Leonardis et al., 2013). However, all of these explanations Toxicology, 29(5), 387–389. rely on blood AA being the source of milk AA. They are therefore Lachenmeier, D. W., & Monakhova, Y. B. (2011). Short-term salivary acetaldehyde increase due to direct exposure to alcoholic beverages as an additional cancer negated by our failure to detect Eth and AA in the blood plasma. risk factor beyond ethanol metabolism. Journal of Experimental & Clinical Cancer Eth is converted in mammalian liver cells into AA by ADH, and Research, 30,3. then AA is further oxidized into , which is harmless, by Lachenmeier, D. W., & Sohnius, E.-M. (2008). The role of acetaldehyde outside ethanol metabolism in the carcinogenicity of alcoholic beverages: Evidence AADH. These two oxidation reactions are coupled with the reduc- from a large chemical survey. Food and Chemical Toxicology, 46(8), 2903–2911. tion of NAD+ to NADH (Fig. 1). In Eth-forming bacteria and , Leitner, G., Krifucks, O., Merin, U., Lavi, Y., & Silanikove, N. (2006). Interactions the last steps of fermentation involve conversion of pyru- between bacteria type, proteolysis of casein and physico-chemical properties of bovine milk. International Dairy Journal, 16(6), 648–654. vate to AA and by the pyruvate decarboxy- Linderborg, K., Salaspuro, M., & Väkeväinen, S. (2011). A single sip of a strong lase, followed by conversion of AA to Eth. The latter reaction is also alcoholic beverage causes exposure to carcinogenic concentrations of catalyzed by ADH, which in this case operates in a direction oppo- acetaldehyde in the oral cavity. Food and Chemical Toxicology, 49(9), 2103–2106. Nieminen, Mikko T., Novak-Frazer, L., Collins, R., Dawsey, S., Dawsey, S., Abnet, C. C., site to that in the mammalian liver. Stress has been shown to ... Rautemaa, R. (2013). Alcohol and acetaldehyde in African fermented milk induce the conversion of mammary gland epithelial cells to aerobic Mursik – A possible etiologic factor for high incidence of in (Silanikove, Merin, Shapiro, & Leitner, 2014; Silanikove Western Kenya. Cancer Epidemiology Biomarkers & Prevention, 22(1), 69–75. et al., 2011). The conversion to aerobic glycolysis is associated with Pontes, H., de Pinho, P. G., Casal, S., Carmo, H., Santos, A., Magalhães, T., et al. (2009). GC determination of , acetaldehyde, ethanol, and in biological upregulation of oxidation to coupled to matrices and cell culture. Journal of Chromatographic Science, 47(4), 272–278. reduction of NADH to NAD+ by lactic dehydrogenase (Silanikove Ramdzan, A. N., Mornane, P. J., McCullough, M. J., Mazurek, W., & Kolev, S. D. (2013). et al., 2011, 2014). It is also associated with increased oxidation Determination of acetaldehyde in saliva by gas-diffusion flow injection analysis. Analytica Chimica Acta, 786, 70–77. of oxaloacetic acid into malic acid coupled with reduction of NADH Redetzki, H. M., & Dees, W. L. (1976). Comparison of four kits for enzymatic to NAD+ by malic dehydrogenase (Silanikove et al., 2011). Our determination of ethanol in blood. Clinical Chemistry, 22(1), 83–86. results seem to show that the Eth–AA axis operates at a low level Schlatter, J., Chiadmi, F., Gandon, V., & Chariot, P. (2014). Simultaneous determination of methanol, acetaldehyde, acetone, and ethanol in human in mammary gland epithelial cells under non-stressful conditions, blood by gas chromatography with flame ionization detection. Human & which may reflect the tendency of mammary gland epithelial cell Experimental Toxicology, 33(1), 74–80. enzymes to work in the reverse direction (substrate reduction – Seitz, H. K., & Stickel, F. (2007). Molecular mechanisms of alcohol-mediated carcinogenesis. Nature Reviews Cancer, 7(8), 599–612. NADH oxidation) under stressful conditions. Though, we have no Shapiro, F., Shamay, A., & Silanikove, N. (2002). Determination of lactose and D- proof for this possibility, nor do we have any alternative galactose using thio-NAD(+) instead of NAD(+). International Dairy Journal, 12 explanation, the methods developed here can serve to resolve this (8), 667–669. Shapiro, F., & Silanikove, N. (2010). Rapid and accurate determination of D- and L- mystery. lactate, lactose and galactose by enzymatic reactions coupled to formation of a fluorochromophore: Applications in food quality control. Food Chemistry, 119 4. Conclusions (2), 829–833. Shapiro, F., & Silanikove, N. (2011). Rapid and accurate determination of malate, citrate, pyruvate and oxaloacetate by enzymatic reactions coupled to formation The described modifications of enzymatic methods for the of a fluorochromophore: Application in colorful and fermentable food determination of Eth and AA considerably increased the existing (yogurt, wine) analysis. Food Chemistry, 129(2), 608–613. Silanikove, N., Merin, U., Shapiro, F., & Leitner, G. (2014). Milk metabolites as methodologies’ sensitivity and reproducibility. The developed indicators of mammary gland functions and milk quality. 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