Food Chemistry 201 (2016) 270–274
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Detecting ethanol and acetaldehyde by simple and ultrasensitive
fluorimetric methods in compound foods
M. Zachut, F. Shapiro, N. Silanikove ⇑
Biology of Lactation Laboratory, Department of Ruminant Sciences, Institute of Animal Sciences, Agricultural Research Organization, Volcani Center, Bet Dagan 50250, Israel
a r t i c l e
i n f o
Article history:
Received 15 July 2015
Received in revised form 20 October 2015
Accepted 19 January 2016
Available online 21 January 2016
Keywords:
Ethanol
Acetaldehyde
Fluorimetric assay
a b s t r a c t
There is a need for simple, accurate, and rapid analysis of ethanol (Eth) and acetaldehyde (AA) in a wide
variety of beverages and foods. A novel enzymatic assay coupled to formation of fluorescent chromophore is presented. Eth detection was further improved by adding semicarbazide to the reaction mixture, which interacts with AA and prevents its inhibitory effect on Eth oxidation. The limits of detection of
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
that with commercial kits (23%). The high sensitivity of the developed method enables detection of AA
in common foods [e.g., bio-yogurt (12.2 mg/L), and the existence of endogenous Eth (1.8 mg/L) and AA
(2.0 mg/L) in bacteria-free non-fermented bovine milk], which could not measured so far by enzymatic
methods.
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
Ethanol (Eth) and acetaldehyde (AA) are metabolites, which are
produced during fermentation processes and are commonly present in fermented beverages and foods. They may therefore be present in humans’ biological fluids. In mammals, AA is the main
product of Eth oxidation in the liver (Hipólito, Sánchez, Polache,
& Granero, 2007). Accurate measurements of Eth and AA are therefore required in a variety of matrices, such as alcoholic beverages,
foodstuffs, cosmetics, and pharmaceuticals. Moreover, measurement of Eth and AA in the blood plasma and other biological fluids
is of particular importance for the diagnosis and treatment of
alcohol-use disorders, as biomarkers for several diseases, in
acute intoxications, and in forensic settings (Schlatter, Chiadmi,
Gandon, & Chariot, 2014).
Many methods, such as gas-diffusion flow-injection analysis
(FIA), electroanalysis, FIA-electroanalytical detection, infrared (IR)
spectroscopy, direct injection gas chromatography (GC)/flame ionization detection (FID), headspace injection GC/FID, highperformance liquid chromatography (HPLC)/Fourier transform
(FT) and others have been developed for analyses of Eth and AA
(Jain & Cravey, 1972a, 1972b; Ramdzan, Mornane, McCullough,
Mazurek, & Kolev, 2013; Schlatter et al., 2014). However, some of
these methods are not sufficiently accurate (e.g., older versions of
GC; Schlatter et al., 2014), some require complex and expensive
⇑ Corresponding author.
E-mail address: [email protected] (N. Silanikove).
http://dx.doi.org/10.1016/j.foodchem.2016.01.079
0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.
instruments (HPLC). GC/FID has been used for the determination
of Eth and AA and in saliva and alcohol beverages without sample
preparation; however, it requires relatively high sample volumes
( 450 ll) and dedicated equipment (Homann et al., 1997;
Linderborg, Salaspuro, & Väkeväinen, 2011). A particular problem
with the GC/FID method is formation of artificial AA due to Eth oxidations, which requires particular procedures to account for the
problem (Fukunaga, Silanaukee, & Eriksson, 1993). Consequently,
the availability of an analytical method that is simple, rapid,
cost-effective and accurate for the determination of Eth and AA is
desirable. AA is a toxic substance, a class 1 carcinogen and mutagenic at concentrations of 50–100 lM (2.2–4.4 mg/L) (Seitz &
Stickel, 2007). Recently, mutagenic levels of AA have been reported
in various foods (Lachenmeier & Sohnius, 2008; Uebelacker &
Lachenmeier, 2011). Although the detection limit of modern GC
methods is sufficient to detect mutagenic levels of AA in foods
(Lachenmeier & Sohnius, 2008; Pontes et al., 2009), this method
is quite cumbersome for routine analyses. Conversely, current
enzymatic analyses, with detection limits above 10 mg/L (Beutler,
1988), are not suitable for detecting mutagenic levels of AA. Thus,
the need for a fast and versatile method to determine low levels of
AA in various beverages and foods is of particular importance.
Enzymatic methods that utilize alcohol dehydrogenase (ADH)
and AA dehydrogenase (AADH) are well-known and frequently
used to analyze Eth and AA in biological specimens (Beutler,
1988; Redetzki & Dees, 1976). ADH oxidizes Eth to AA and AADH
oxidizes AA to acetic acid. Both enzymes use nicotinamide adenine
dinucleotide (NAD+) as coenzyme, which is reduced during the
M. Zachut et al. / Food Chemistry 201 (2016) 270–274
reaction to form NADH. NADH formation is stoichiometrically
linked to the oxidation of Eth and AA. Thus, the concentration of
NADH in specimens can be used to monitor the concentrations of
metabolites formed by NAD+ dependent dehydrogenases spectrophotometrically or fluorometrically (Beutler, 1988; Redetzki &
Dees, 1976; Shapiro, Shamay, & Silanikove, 2002). However, measuring NADH in foods and other matrices is frequently difficult
and problematic because: (i) these substances contain fat droplets
of varying sizes that scatter light in an unpredictable way; (ii) as a
result of their opaque and colloidal properties, they scatter and
absorb light; (iii) they frequently contain intense colorants that
interfere with the monochromatic absorbance. NADH can be determined by fluorometric means, which are free of these limitations.
However, because additional indigenous biological substances
emit light in the same range, fluorometric determination of NADH
is associated with considerable background noise, which reduces
the sensitivity of the method (Shapiro & Silanikove, 2010, 2011;
Shapiro et al., 2002).
A general solution for measuring metabolites that are involved
in reactions of NAD+-coupled dehydrogenases is to combine the
reaction to another set of coupling reactions: diaphorase (EC
1.6.99.1) oxidizes NADH to NAD+, and this reaction can be coupled
to the conversion of non-fluorometric resazurin to the highly fluorochromophoric substance resorufin (Shapiro & Silanikove, 2010,
2011; Shapiro et al., 2002). To date, this methodology has been
found useful for accurate determination of D- and L-lactate, lactose,
galactose citrate, malate pyruvate and oxaloacetate in milk,
yogurts and colored drinks, such as red wine and beer, without
the need for pretreatments (Shapiro & Silanikove, 2010, 2011).
Oxidation of Eth is particularly sensitive to inhibition by its product, AA (Kristoffersen, Skuterud, Larssen, Skurtveit, & SmithKielland, 2005; Kristoffersen & Smith-Kielland, 2005). A potential
solution to this problem is to force the reaction toward completion,
thereby overcoming product inhibition. When semicarbazide was
added to a reaction solution containing AADH and NAD+, it reacted
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 spectrophotometrically, suggesting that the sensitivity and range of biological
sources without sample preparation might be improved by applying the fluorometric determination of resorufin, as already noted.
Hence, the objective of this study was to apply the abovedescribed modifications to improve the detection of Eth and AA
in compound beverages and foods.
271
2.3. Reaction mixtures
All reagent solutions were prepared fresh once a week with
double-distilled water. For Eth and AA determinations according to
Option 1 (Fig. 1), the reaction mixture was composed of 1 mM
NAD+, 48 lM resazurin, 1U/mL, diaphorase, 100 mM KCl, 0.0004%
(w/v) Triton X-100, and 50 lL ADH (10 kU/mL) and 10 lL AADH
(75 U/mL) dissolved in 50 mM potassium phosphate buffer, pH 7.6.
For the separate determination of AA, addition of ADH was omitted .
In Option 2 (Fig. 1), the stock solution for AA was prepared without
ADH and contained 75 mM semicarbazide and 130 mM glycine.
2.4. Standard curves
Stock solutions were prepared by dissolving 100 mg/mL of Eth
or AA in double-distilled water. Standards were prepared by serially diluting the stock solutions of the test substances in distilled
water to yield concentrations of 1, 2.5, 5, 10, 25, 50, 100, 250,
500 and 1000 mg/L.
2.5. Reaction procedures
All procedures were carried out in the wells of a 96-well microplate suitable for fluorometric reading. Reaction mixture (100 lL)
and test solution (10 lL) (standard or test samples) were incubated
together in the wells for 30 min at room temperature. The plates
were read in a fluorometer (ELx800, BioTek Instruments, Winooski,
VT, USA) at excitation and emission wavelengths of 540 and
590 nm, respectively.
2.6. Biological and food samples
Milk was sampled from the commingled milk of six cows with
bacteria-free udders. Bacteria-free samples were defined as the
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 Pseudomonas fluorescens (100 U/L), AA, Eth (AA and Eth standards are
stored in ampoules), glycine, KCl, b-NAD+ hydrate, 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.
2.2. Assay principle
The enzymatic reactions that served as the basis for the analysis
of Eth and AA and a schematic representation of the reaction
cycling are shown in Fig. 1.
Fig. 1. Schematic representation of assay principles for the determination of
ethanol (A) and acetaldehyde (B). Footnote: in the conventional mode (Option 1),
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
of ethanol is calculated as the difference between (A + B) – B. In the modified mode
(Option 2), ethanol and acetaldehyde are determined separately as described in
panels (A) and (B).
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M. Zachut et al. / Food Chemistry 201 (2016) 270–274
absence of bacterial identification over three samplings taken once
every 3–4 weeks. The sampling procedure and bacterial identification were carried out according to internationally recognized standards (Leitner, Krifucks, Merin, Lavi, & Silanikove, 2006). The milk
sampled for chemical analysis was stored on ice, skimmed
(Silanikove & Shapiro, 2007) and analyzed as described below.
Blood was taken from these cows into heparinized tubes and the
plasma was separated by centrifugation.
In addition, the concentrations of Eth and AA were determined in
the following commercial sources: kefir (locally produced), bioyogurt (fermented milk with addition of probiotic; locally produced,
international brand), beer (lager, locally produced, international
brand), and cognac (alcoholic drink, produced in France). In these
products, the concentrations of Eth and AA were also determined
by commercial kits. Additional commercial sources that were analyzed for Eth and AA concentration were: wine (Merlot, local brand),
vodka (produced in Russia), synthesized vinegar (locally produced),
vinegar from apple juice fermentation (locally produced), balsamic
vinegar (produced in Italy), lemon juice (locally produced), cola soft
drink (locally produced, international brand), and an energy drink
(locally produced, international brand).
For the kefir and bio-yogurt, fluid was extracted by centrifugation and used for analysis. For the other sources, the only preparation was dilution to fit the linear portion of the standard curves.
As a precautionary measure against the possibility of false
results, we also performed a recovery analysis of spiked Eth and
AA by applying the levels found in the milk and carrying out the
standard curve analysis (Option 2 in the case of Eth) using milk
as the medium.
2.7. Calculation, validation parameters and statistical analysis
The concentrations of the analyzed metabolites were derived
from linear regression analysis of the calibration curves. For the
determination of linearity, regression lines were calculated as
y = a + bx, where x was concentration, and y the response. Ten concentration points in triplicate were used to prepare the calibration
curves. For each compound, the coefficient of determination (R2)
was calculated and the repeatability was assessed based on the relative standard deviation (RSD) values for the corresponding
response factors. A blank was run in five replicates and its values
were subtracted from the readings. The limit of detection (LOD)
for each metabolite was determined by calculating the y value
(concentration) derived from blank + 3 SD of the blank. The limit
of quantification (LOQ) was derived from the blank + 10 SD of the
blank. In the case of milk, we used the intercept to calculate LOD
and LOQ. Repeatability of the assay method was analyzed by calculating the RSD values of three replications of the standard curve
analysis. The upper limit of linearity was determined when the
expected response (based on the linear regression of lower concentration points) was smaller than expected by 3 RSD. Day-to-day
repeatability was estimated by calculating the RSD derived from
analyses of the standard curve over 3 consecutive days. Recovery
was determined using an added external standard. The samples
were spiked at two levels (1 and 10 lM), each in triplicate with
known quantities of the test compound, and the percentage recovery was calculated. The percentage of recovery rate for the tested
compounds was established from the experimental response values [(blank + standard) blank] obtained according to the calibration curves and the real concentration of the standard added. Each
of the foods was analyzed in triplicate, each time on 3 separate
days. Within-day and between-day repeatability was defined as
the SD of the respective measurements and presented as SD as percentage of the mean (RSD). Statistical differences between values
of the same sample measured by different methods were obtained
by paired t-test.
3. Results and discussion
3.1. The standard curves
Determination of Eth using semicarbazide in a separate reaction
(Option 2) proved advantageous compared to the combined reaction (Option 1) in terms of higher R2 of the calibration curve, lower
LOD and greater linear range of the reactions (Represented by
upper limit of detection, ULD, in Table 1), which was at least twice
as large with Option 2 (Table 1). In general, the improved LOD, LOQ
and range of linearity obtained using Option 2 was consistent with
similar improvement attained by converting the colorimetric
detection of NADH to fluorometric methods (Shapiro &
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
were markedly better than those obtained with currently available
enzymatic methods (Beutler, 1988; Redetzki & Dees, 1976). Thus,
our method enables the detection of Eth at the level required for
forensic settings and the reliable detection of mutagenic levels of
AA in foods (Tables 2 and 3).
3.2. Comparison of developed method and commercial kits
We compared Eth and AA concentrations in commercial beverages and foods as analyzed by commercial kits for Eth and AA, by
Options 1 and 2 for Eth, and by the developed fluorescent method
for AA (Table 2). The commercial kit for Eth determination worked
on the same principle as our method using Option 1, although the
manufacturer did not disclose the reagents used, including those
for the fluorescent coupling reaction. The commercial kit used for
AA was a colorimetric version of the method developed here.
Within each compared source, there was no significant difference
between either Eth or AA concentrations determined by the different methods. The concentration of AA in the bio-yogurt was three
times higher than the concentration of 100 lM AA (4.4 mg/L)
which is considered to be mutagenic (Lachenmeier & Sohnius,
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.
a
b
Substance
R2
LOD (mg/L)
LOQ (mg/L)
ULD (mg/L)
RSDb of the estimate (%)
Day-to-day RSD (%)
Ethanol, Option 1
Ethanol, Option 2
Ethanol, Option 2, in milk
Acetaldehyde
Acetaldehyde in milk
0.991
0.999
0.998
0.998
0.997
0.6
0.5
0.5
0.9
1.0
2.0
1.6
1.7
3.1
3.2
50
100
100
100
100
5
3
4
4
5
7
4
4
5
6
Same measurements over 3 days.
RSD – relative standard deviation.
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M. Zachut et al. / Food Chemistry 201 (2016) 270–274
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 2
Acetaldehyde by kit
Acetaldehyde
Source of sample
Concentration (mg/L)
RSD (%)
Beer (lager, international brand)
41120 (4.1%, w/v)
41750 (4.2%, w/v)
21.2
41.4
Ethanol Option 1
41450 (4.1%, w/v)
5.7
0.1
35.2
0.1
12.5
Source of sample
Concentration (mg/L)
RSD (%)
Cognac (from France)
456750 (45.7%, w/v)
28.2
452980 (45.3%, w/v)
9.1
0.7
18.1
0.7
5.9
Source of sample
Concentration (mg/L)
RSD (%)
Kefir (fermented milk, local brand)
3455
3540
21.1
17.0
3340
9.0
1.4
10.3
1.4
23.1
Source of sample
Concentration (mg/L)
RSD (%)
Average RSD (%)
Bio-yogurt (yogurt with probiotic culture, international brand)
25.2
22.6
27.5
25.1
17.8
12.2
23.9
28.6
9.0
13.8
28.6
23.0
12.2
9.0
12.6
450315 (45.0%, w/v)
38.2
Table 3
Concentrations of ethanol and acetaldehyde (lg/ml) in cow blood plasma, cow fresh
milk and a variety of beverages and foods.
Source of sample
Ethanol
(mg/L)
RSD
(%)
Acetaldehyde
(mg/L)
RSD
(%)
Cow plasma
Not
detected
1.8
136020
(13.6%,
w/v)
665040
(66.5%,
w/v)
103.8
–
Not detected
–
8.0
1.3
2.0
11 (250 lM)
7.0
6.6
1.1
5.5 (125 lM)
12.5
2.8
8.1 (183 lM)
14.3
179.2
2.2
60.0
8.0
69.3
3.1
166.0
2.0
0. 6
25.5
1.8
10.9
7.3
1.8
4.7
15.7
0.9
1.7
10.3
3.9
Cow milk, raw, fresh
Wine, Merlot, local brand
Vodka, Russian brand
Vinegar, synthesized, local
brand
Vinegar, fermented from apple
juice, local brand
Vinegar, fermented, balsamictype, Italy
Lemon juice, local brand (no
additions)
Cola soft drink
Energy drink, international
brand
The concentrations of Eth in beer and cognac were consistent
with the expected levels (Lachenmeier & Sohnius, 2008) and were
within ±10% of those declared by the producer. However, the
pooled RSD (within and between days) of Eth ranged between 4%
and 12%, 9% on average, and was considerably lower than that
obtained with Option 1 (17–41%, average 18%) or the commercial
kit (21–25%, average 24%). Similarly, the RSD of AA concentration
determined by the fluorescent method ranged from 6 to 23%, 9%
on average, which was much lower than the values obtained
with the commercial kit (10–35% and 23% on average). It could
be concluded that using Option 2 for the Eth determination and
the developed method for AA offers a considerable improvement
in repeatability over the methods to which they were
compared, and thus increased assurance that the observed values
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
similar modifications have been made for the determination of various metabolites (Shapiro & Silanikove, 2010, 2011).
3.3. Mutagenic levels of AA in some common alcoholic drinks, foods
and food supplements
Concentrations of Eth by Option 2 and AA in a range of beverages and foods are presented in Table 3. As noted in Table 2, Eth
levels in alcoholic drinks were consistent with expected levels
and the values indicated by the producers on the labels. No significant levels of Eth or AA were found in the lemon juice, cola soft
drink or energy drink, all non-fermented products. In contrast,
except in the tested varieties of beer and kefir (Table 2), all tested
fermented products—bio-yogurt (Table 2), wine, vodka and vinegars (Table 3)—contained mutagenic levels of AA. The millimolar
levels of AA in apple vinegar and balsamic vinegar, which are
world-famous for their taste, were 14- and 34-fold higher than
mutagenic levels (Seitz & Stickel, 2007) of this compound (Table 3).
In a close note, we would like to remark that further research is
required to correlate between in vitro finding on mutagenic levels
of AA in foods and carcinogenicity. However, formation of DNA
adducts from AA (Brooks & Theruvathu, 2005), the prevalence of
DNA adducts in the oral cavity, in association with alcohol drinking
(Balbo et al., 2012) and alcohol associated increases risk of cancer
of oral cavity and pharynx, esophagus (Bagnardi et al., 2015;
Lachenmeier & Monakhova, 2011) and high incidence of esophageal cancer in African population consuming fermented milk with
high content of AA (Nieminen et al., 2013) strongly suggest that AA
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 gut. Our restricted survey is consistent with a larger one that
showed that many common drinks and foods may contain mutagenic levels of AA (Lachenmeier & Sohnius, 2008; Uebelacker &
Lachenmeier, 2011). As applied by Uebelacker and Lachenmeier
(2011), a digestion step with simulated gastric fluid may be
required to account for AA bound to proteins or other molecules
in food samples. In conclusion, research of the type described in
Lachenmeier and Sohnius (2008) and Uebelacker and
Lachenmeier (2011) papers may lead to improved food security
by convincing regulatory bodies to adjust regulatory roles to findings; for instance, by preventing the addition of pure forms of AA to
foods. The method developed here provides a simple, accurate and
practical means of gaining broad information on AA content in
foods.
3.4. Concentrations of Eth and AA in non-fermented bovine milk
No detectable levels of Eth or AA were found in the cows’ blood
plasma (Table 3). However, the fresh milk, which was sampled
from cows that were free of bacterial infections, contained low
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 &
Singh, 2007). Recovery levels of Eth and AA relative to the level
found in milk were 98 ± 3% and 91 ± 6%, respectively. LOD and
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M. Zachut et al. / Food Chemistry 201 (2016) 270–274
LOQ of standard curves made in milk did not differ significantly
from those made in water (Table 1).
To the best of our knowledge, this is the first time that Eth has
been detected in bacteria-free and non-fermented mammal’s milk.
This might be related to the significant improvement in its detection levels. The AA levels were higher than the LOD and were about
two-thirds of the LOQ and therefore, this result most likely represents the actual content of AA in the milk. Furthermore, AA has
been recently detected in unfermented raw milk of cow, buffalo,
goat and sheep (De Leonardis, Lopez, Nag, & Macciola, 2013),
although we believe that the reported levels (20–65 mg/L) in that
case were about 10-fold higher than the actual ones.
The content of AA in milk might have been related to the transfer of AA from the air to the bloodstream upon inhalation, further
passing into the milk (De Leonardis et al., 2013). Additional possible sources for AA in the milk are via fodder digestion and absorption (De Leonardis et al., 2013). However, all of these explanations
rely on blood AA being the source of milk AA. They are therefore
negated by our failure to detect Eth and AA in the blood plasma.
Eth is converted in mammalian liver cells into AA by ADH, and
then AA is further oxidized into acetic acid, which is harmless, by
AADH. These two oxidation reactions are coupled with the reduction of NAD+ to NADH (Fig. 1). In Eth-forming bacteria and yeast,
the last steps of glucose fermentation involve conversion of pyruvate to AA and carbon dioxide by the enzyme pyruvate decarboxylase, followed by conversion of AA to Eth. The latter reaction is also
catalyzed by ADH, which in this case operates in a direction opposite to that in the mammalian liver. Stress has been shown to
induce the conversion of mammary gland epithelial cells to aerobic
glycolysis (Silanikove, Merin, Shapiro, & Leitner, 2014; Silanikove
et al., 2011). The conversion to aerobic glycolysis is associated with
upregulation of pyruvic acid oxidation to lactic acid coupled to
reduction of NADH to NAD+ by lactic dehydrogenase (Silanikove
et al., 2011, 2014). It is also associated with increased oxidation
of oxaloacetic acid into malic acid coupled with reduction of NADH
to NAD+ by malic dehydrogenase (Silanikove et al., 2011). Our
results seem to show that the Eth–AA axis operates at a low level
in mammary gland epithelial cells under non-stressful conditions,
which may reflect the tendency of mammary gland epithelial cell
enzymes to work in the reverse direction (substrate reduction –
NADH oxidation) under stressful conditions. Though, we have no
concrete proof for this possibility, nor do we have any alternative
explanation, the methods developed here can serve to resolve this
mystery.
4. Conclusions
The described modifications of enzymatic methods for the
determination of Eth and AA considerably increased the existing
methodologies’ sensitivity and reproducibility. The developed
methods were simple, accurate and versatile, and enabled determining Eth and AA levels with no special sample preparation in a
wide variety of beverages, foods and biological specimens.
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