Assessment of Alcohol Production by Uncoupling Oxidative

Phosphorylation on Some Pollutants

OLGA PINTILIE1, MARIUS ZAHARIA2*, ADELINA COSMA2, MANUELA MURARIU3, ROBERT GRADINARU2, GHEORGHE G. BALAN4,
GABI DROCHIOIU2, ION SANDU1,5*
1
Al. I. Cuza University of Iasi, Department of Geography and Geology, 11 Carol I Blvd., 700506, Iasi, Romania
2
Al. I. Cuza University of Iasi, Department of Chemistry, 11 Carol I Blvd., 700506, Iasi, Romania,
3
Petru Poni Institute of Macromolecular Chemistry, 41A Gr. Ghica Voda Alley, 700487, Iasi, Romania
4
Grigore T. Popa University of Medicine and Pharmacy, Faculty of Dental Medicine, 16 Universitatii Str., 700115, Iasi, Romania
5
ARHEOINVEST Interdisciplinary Platform, Laboratory of Scientific Investigation and Conservation of Cultural Heritage, Al. I.
Cuza University of Iasi, 22 Carol I, 700506, Iasi, Romania

Dinitrophenol pesticides are major environment pollutants. Today, DNP is used by bodybuilders, often illegally,
to rapidly lose body fat before contests. It is thought that they uncouple the oxidative phosphorylation by
carrying protons across the mitochondrial membrane, leading to a rapid consumption of energy without
generation of ATP. Uncoupling agents such as dinitrophenols determine a switch from respiration mode to
a fermentative one. As a consequence, lactic acid production is increased in animal cells, whereas plant
and yeast cells adopt a biochemical pathway leading to alcohol. We propose here a very simple and
effective method to show uncoupling properties of some chemicals based on alcohol determination in
uncoupler-treated organisms. The feasibility of our method, which is based on Cr6+ reaction with alcohols is
investigated. Analytical parameters of Cr6+ absorption at 350 nm and that of Cr3+ ions at around 600 nm are
studied.
Keywords: dinitrophenol pesticides, uncoupling agent, alcohol determination, yeast, oxidative
phosphorylation

In spite of their toxicity to the environment, many

dinitrophenols and dinitrophenyl derivatives are still in use
as herbicides, insecticides or even therapeutic agents [14]. Sub-acute poisoning is associated with weight-loss and
the substance had been prescribed for this purpose during
the 1930s in the United States before being banned due to
serious side effects [5-10]. Numerous pesticides act as
uncoupling agents of oxidative phosphorylation at low
concentrations [11,12], and inhibit the electron transport
chains, probably before cytochrome c at higher
concentrations [13, 14]. Dinitrophenol disrupts the H+
gradient reducing ATP synthesis [15,16]. The permeability
of mitochondrial membranes to protons is increased by
the conversion failure from ADP to ATP [11,17,18]. Under
these conditions, much of our food that we eat could not
be used for ATP synthesis are we lose weight. However,
too much inhibitor and we could make too little ATP for
life. The difference between weight loss and death is only
a small concentration change in dinitrophenol, making the
drug dangerous [19, 20]. According to most hypotheses, it
uncouples oxidative phosphorylation by carrying protons
across the mitochondrial membrane, leading to a rapid
consumption of energy without ATP generation. The level
of toxicity of dinitrophenyl derivatives is closely related to
ATP inhibition even in the presence of oxygen, leading to
alcohol formation in living cells of some biological materials
[21, 22].
Yeast is a microorganism which produces ATP both
aerobic and anaerobic, and therefore are ideal living bodies
to investigate the shift from cell respiration to fermentation.
Also is used in baking carry out the alcoholic fermentation
[23]; the CO2 produced by pyruvate decarboxylation causes
bread to rise, and the ethanol produced evaporates during

baking. Higher plants also make alcohol by the same

reaction pathway [24]. Alcohol determination is currently
carried out with enzymatic methods [25].
Recently, we patented a method for identifying the
uncoupling agents of oxidative phosphorylation, as well
as quantifying the intensity of their action by determining
the ethanol produced in living cells of some biological
materials. Under the action of uncoupling agents,
dinitrophenols and other compounds, alcohol is liberated,
which can be colorimetrically determined by the reduction
of the orange colored potassium dichromate (K2Cr2O7) to
green colored salts of chromium (III), in the presence of
sulfuric acid (H2SO4). A well-defined volume of sample is
taken from the green colored solution, diluted with distilled
water, and its absorbance measured in a spectrophotometer or colorimeter. The calibration curve is carried
out with different concentrations of ethanol [26].
Experimental part
Reagents. All reagents were purchased from Merck
(Darmstadt, Germany) and used without further
purification. Dinitrophenols were acquired from SigmaAldrich (SUA). The used reagents were of analytical grade,
and the aqueous solutions were prepared with double
distilled high purity water (R = 18.2 ).
Biological materials. Bakers yeast was purchased
weekly from SC Rompak srl Pascani (Romania), and kept
in a humidor at 4 C.
Instruments. Spectrophotometer UV-Vis model Libbra
S35 PC UV/VIS ((Biochrom, UK) equipped with quartz
cuvettes with optical length of 1 cm.
Protocols. Alcohol monitoring assay: 5 g yeast and 5 g
D-glucose were dissolved in 100 mL distilled. In the pot
hole center of Conway dish 1 mL yeast suspension was

* email: zaharia.marius2011@yahoo.com; ion.sandu@uaic.ro

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375

mixed with 1 mL NaOH (0.1N). In the outlet 3 mL K2Cr2O7

10-2 M and 3 mL H2SO4 96% were added. Conway dish was
incubated on a hotplate at a temperature of 55 0C for
different time intervals. After reaction the outlet content
was diluted to 10 mL final volume. The absorption spectra
of resulted solution were recorded on a Libra S35 PC UVVIS spectrophotometer using 1-cm quartz cuvettes, within
the range of = 250-650 nm. Measurements were made
with different amounts of suspended yeast. Yeast can be
replaced by living plant materials as well.
Results and discussions
In figure 1, representing the calibration of the method of
work, shows the following: the 10-2 M solution of K2Cr2O7
displayed four absorbance maxima at 350 nm with an
intensity of 0.450; treatment with distilled water (control)
the following absorbance values were measured was
0.269, in this case was not highlighted a predetermined
reduction of potassium dichromate. The 10-2 M ions solution
of Cr3+ , the green color, used for highlighting the reduction
process of potassium dichromate, the following values
were measured: A= 0.082 (296 nm), A= 0.061 (403 nm)
and A= 0.049 (573 nm), respectively. The resulting solution
by treating the yeast with a 50 M solution of 2,4-DNP has
displays a absorption maxima at 350 nm (A= 0.014),
indicating the ethnaol formation which has reduced
dichromate potassium salts of chromium trivalent, so the
green colored final solution contains Cr2(SO4)3.
In this example, the 10-2 M solution of Cr3+ has been
used to highlight the spectral changes in the case of the
entire quantity of potassium dichromate should be reduced.
However, can be use 50 M 2,4-DNP as standard, in
establishing uncoupling activity of some another pollutants.
In parallel, carry out a calibration curve using ethanol
(in the range of concentrations 0-5 mm), in order to
determine the exact concentration of alcohol is formed
under the action of the uncoupling agents (fig. 2).
In the presence of yeast, the absorption spectrum of
potassium dichromate has the same ultraviolet bands as
for pure K2Cr2O7 solution (fig. 3). 2,4-dinitrophenol (2,4DNP) mislead the passage of yeasts from aerobic

Fig. 1. Calibration method using aqueous acid solutions by UV-Vis

spectroscopy.

Fig. 2. Calibration curve with different concentration of alcohol

376

Fig. 3. UV spectra of 2,4-dinitrophenol, potassium dichromate and

Cr(III) ions in the presence of yeast suspensions

respiration to anaerobic fermentation and alcohol formation

which enable to reduce Cr6+ ions at Cr3+ ions, respectively
the appearance of the green color correlated with the
characteristic band disappearance of potassium
dichromate. Also, according with these data experimental
we can say that: the concentration or the amount of alcohol
produced is proportional with the uncoupling action of
oxidative phosphorylation intensity. This assertion can be
placed in the highlighted on the basis of spectrophotometer
measurements both in the ultraviolet, at approximately 350
nm by reducing the intensity of the absorption maxima, as
well as in the visible, at approximately 600 nm, where
growth of the maximum intensity of the absorption who is
directly proportional with the ions concentration of trivalent
chromium formed in the presence of alcohol.
Several dinitrophenol derivatives were tested within the
germination experiments comparatively to some
dinitrophenyl ethers in order to better understand the
toxicity mechanism [27]. Both ethers and phenol
derivatives displayed a similar pattern of biological activity
inhibiting seed germination, most probably by blocking
oxidative phosphorylation. A novel mechanism of action
of these pesticides was proposed, which is concordant
with Macovschis biostructural theory and not with proton
translocation hypothesis by Peter Mitchell.
The mechanism of biological activity as well as that of
toxicity of dinitrophenyl derivatives with uncoupling activity
might be related to energy transfer in ATP formation and
not with proton translocation through the mitochondrial
membranes [28]. A more complex toxicity mechanism is
needed to explain the specificity of dinitrophenyl ethers
when compared to simple dinitrophenols. Conformational
changes of enzymes involved in respiratory processes by
such compounds have to be studied as well.
Conclusions
This paper related a method for identifying the
uncoupling agents of oxidative phosphorylation, as well
as quantifying the intensity of their action by determining
the ethanol produced in living cells of some biological
materials. Thus, the action of uncoupling agents, such as
dinitrophenols, results in the inhibition of respiration of
eukaryotic cells whereas the alcohol formed can be
colorimetrically determined by the reduction in the
presence of sulfuric acid (H2SO4) of the orange colored
potassium dichromate (K2Cr2O7) to green colored salts of
chromium (III).
Second, the micro-method was applied to characterize
various uncoupling agents, and its analytical parameters
have been studied. This micro-method was tested using
dinitrophenols and related compounds; the method is
reproductible, sensitive and selective. It can be used to

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REV.CHIM.(Bucharest) 67 No.2 2016

test new compounds in order to discovery new properties

of chemical compounds based on alcohol production by
fermentation. Further investigations should be undertaken
to clarify these obscure aspects of dinitrophenols biological
activity.
Acknowledgments : Funding from the Romanian Government (PN-IIPT-PCCA-2013-4-1149, Contract 107/2014) is gratefully acknowledged.
Marius Z. acknowledges the Grants for young researchers of UAIC
(Contract 21754/03.12.2015) project from Alexandru Ioan Cuza
University of Iasi.

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