international ozone association

Ozonized Unsaturated Triglycerides as Precursors of
Urinary Dicarboxylic Acids
Daniel Jardines, Oscar Ledea and Zullyt Zamora
Ozone Research Center, National Center for Scientific Research,
Ave 230 y 15, Playa, P.O. Box 6414, Havana City, Cuba.
FAX: 21-0233, e. mail: [email protected]
Abstract
Oral administration of ozonized sunflower oil to Wistar rats has produced
changes in the urinary content of dicarboxylic acids. Heptanedioic acid (Pimelic
acid) and nonanedioic acid (Azelaic acid) were the major increased dicarboxylic
acids founded. The aim of this work is the study of the urinary dicarboxylic acid
profiles of Wistar rats, orally treated with ozonized standard triglycerides. The
dicarboxylic acids were extracted and derivatized before the analysis by Gas
Chromatography – Mass Spectrometry. The urinary dicarboxylic acid profiles of
the rats that received ozonized triolein, only showed the increasing of
heptanedioic and nonanedioic acids. However, when ozonized trilinolein was
applied, the profile is similar to that obtained, when ozonized sunflower oil was
administered. A biochemical mechanism to explain the formation of dicarboxylic
acids from ozonated unsaturated triglycerides was proposed.
Introduction
Ozonized Sunflower Oil (OLEOZON®) is a registered drug that has shown
antimicrobial effects against virus, bacteria and fungi (Cajigas et al., 1990; Lezcano et
al., 1996; Lezcano et al., 1998). On the other hand, toxicological studies of
OLEOZON® have demonstrated that this product is not mutagenic or genotoxic and
has not secondary reactions in human patients (Llerena et al., 1995; Martinez et al.,
1995; Remigio et al., 1998). Since 1994 it has being studied the use of ozonated
sunflower oil in the treatment of giardiasis in animal models and in humans
(Menéndez et al., 1995; Hernández, 1999).
Oral administration of ozonized sunflower oil to Wistar rats has produced changes in the
urinary content of dicarboxylic organic acids (Jardines et al., 1998). An increment in urine of
heptanedioic, octanedioic, octenedioic, nonanedioic, decenedioic and dodecenedioic acids has
been observed. Heptanedioic acid (Pimelic acid) and nonanedioic acid (Azelaic acid) were the
major increased dicarboxylic acids founded.
It is supposed that these increased acids are produced because of the unsaturated fatty acid
metabolism and a possible mechanism to explain it, was previously proposed (Fig. 1)
(Jardines et al., 1998). Linoleic and oleic acids are the main unsaturated fatty acids present in
the sunflower oil (Vajda and Saenz, 1976). These fatty acids have two and one double bonds
respectively in their structure and that’s why they quickly react with the ozone.
The reaction between ozone and unsaturated triglycerides occurs by the well-known Criegee
mechanism (Bailey, 1978). Taking into consideration the unsaturated fatty acid composition
of sunflower oil and the ozone-olefin reaction mechanism, during the ozonation of
unsaturated triglycerides, aldehydes and carboxylic acids with three, six and nine carbon
atoms are expected to be obtained. In this reaction hydroperoxides, ozonides and some others
peroxidic or polyperoxidic species could also be obtained (Ledea O. et al, 1998). The
peroxidic and hydroperoxidic species partially decompose forming again aldehydes and
carboxylic acids with different carbon lengths in their structures (Ledea O. et al, 2001).
All the ozonation triglycerides products are metabolized in the organism by the action of
different enzymes (lipase, aldehyde dehydrogenase, peroxidase and glutation-S-transferase)
and they are converted to different dicarboxylic acids (final excretion products). One of the
carboxylic groups is obtained by the action of the lipase and the other by the combination of
ozonation and some enzyme action (aldehyde dehydrogenase, peroxidase and glutation-Stransferase).
The aim of this work was the study of the urinary dicarboxylic acid profiles of Wistar rats,
orally treated with ozonized standard triglycerides (Triolein and Trilinolein) using a
combination of a liquid – liquid extraction method with the Gas Chromatography / Mass
Spectrometry techniques.
O3
II
I O3
R1-CO 2-CH 2
CH 3-(CH 2)4-CH= CH-CH 2-CH= CH-(CH 2)7-CO 2-CH
O3
R3-CO 2-CH 2
I
CH 3-(CH 2)7-CH= CH-(CH 2)7-CO 2-CH
R2-CO 2-CH 2
Linoleil-diacilglyceride
R1-CO 2-CH 2
R4-CO 2-CH 2 O leil-diacilglyceride
R3-CO 2-CH 2
G 3-5 -C 10H 18-CO 2-CH
G 1-4 -(CH 2)7-CO 2-CH
R2-CO 2-CH 2
R4-CO 2-CH 2
Lipase
Lipase
G 3-5 -C 10H 18-CO 2H
O HC-CH 2-CH= CH-(CH 2)7-CO 2H
G 1-4 -(CH 2)7-CO 2H
12-oxododecenoic acid
Aldehyde dehyidrogenase
+
9-oxononanoic acid
Aldehyde dehyidrogenase
+
H O 2 C -(C H 2 )7 -C O 2 H
H O 2 C -C H 2 -C H = C H -(C H 2 )7 -C O 2 H
β−oxidation
O HC-(CH 2)7-CO 2H
3-d od ecened ioic acid
A zelaic acid
H O 2 C -(C H 2 )5 -C O 2 H
β−oxidation P im elic acid
H O 2 C -C H 2 -C H = C H -(C H 2 )5 -C O 2 H
3-d ecened ioic acid
β−oxidation
β−oxidation
H O 2 C -(C H 2 )6 -C O 2 H
O ctaned ioic acid
H O 2 C -C H 2 -C H = C H -(C H 2 )3 -C O 2 H
3-octened ioic acid
P rocess Product
G
O
1
C 8H 17-CH
CH- Reduction
O HCO HC-
CH- Reduction
O HCO HC-
O O
O
2
C 8H 15-CH
O O
3
O HC-
O xidation
HO 2C-
HO O CH- O xidation
OH
O
CH- Reduction
5 C 5H 11-CH
O O
HO 2C-
4
O HCO HC-
Figure 1. Proposed mechanism for urinary dicarboxylic acid formation in Wistar rats after oral administration of
ozonated sunflower oil.
Materials and Methods
Solvents and Reagents
Analytical grade diethyl ether, ethyl acetate, methanol, anhydrous sodium sulfate, sodium
chloride, potassium hydroxide, and hydrochloric acid were obtained from BDH (England). Nnitro-N-metil-p-toluensulfonamide, trilinolein (99%), and triolein (99%) were purchased from
Sigma (USA). All the reagents were used without previous purification.
Unsaturated Triglycerides Ozonation
Fifty milliliters of the substrate was placed in a bubbling reactor, with an oxygen flow of
10 L/h. The reactor was immersed in a water bath at 25 oC. It was used an OZOMED-400
ozone generator (Cuba). The ozone dose was determined by measuring the absorbance at 256
nm, in an Ultrospec III spectrophotometer (Pharmacia). The applied ozone dose was between
50 - 55 mg ozone / g of substrate.
Animals
Twenty four female rats Wistar with a corporal weight from 200 to 225 g were placed in
metabolic cages (Tecniplast, Italy) under controlled conditions of temperature and humidity,
water ad libitum and appropriate standard feeding. A group of six animals were orally treated
with a unique dose of 3.3 mL of ozonated triolein by kg of animal’s weight. The other six
animals were treated with the same dose of ozonated trilinolein. Two controls groups of six
animals each were used with the same doses of triolein and trilinolein respectively. The urine
of each animal was collected during 24 hours and kept at -10 oC until they were analyzed.
Creatinine Determination
The Jaffé method was employed for creatinine determination. One milliliter of urine was
diluted with 49 mL of bidestillated water. A small fraction (0.2 mL) of this sample is mixed
in a glass cuvette with 2 mL of a solution containing picric acid (35 mmol/L) and sodium
hydroxide (0.32 mol/L), followed by the absorbance measurement against air at 490
nanometers (Boehringer Mannheim GmbH, 1979).
Liquid - liquid Extraction of Urinary Organic Acids
Urine samples (5 mL), containing appropriate amounts of internal standard (Heptadecanoic
acid) and sodium chloride, were acidulated with hydrochloric acid (pH=1) and extracted two
times. Firstly with ethyl ether and later with ethyl acetate, equal volume of both solvents were
used (5 mL). The organic faces were mixed and dried with anhydrous sodium sulfate. The
mixture was filtered and the solvents were removed under nitrogen flow at room temperature.
One milliliter of ethyl ether was used to dissolve the extracted residue and diazomethane was
bubbled to obtain the methyl esters of the existing dicarboxylic acids. Nitrogen was bubbled
until dryness, and 50 µL of ethyl acetate were used to dissolve the final products. The samples
were kept at -20 oC until the Gas Chromatography / Mass Spectrometry analysis.
Gas Chromatography / Mass Spectrometry Analysis
An AUTOMASS GC/MS (UNICAM, England) was used. A FFAP Supelco capillary column
(30 m, 0.32 mm i.d., 0.25 µm film thickness) was employed for the separation of dicarboxylic
acids methyl esters. The temperature - programming columns conditions used were: 80 oC
initial temperature (2 min), 8 oC/min to 220 oC, and held at 220 oC for 10 minutes. An
ionization voltage of 70 eV over the mass range of 30 - 400 AMU was used to fragment the
components. The interface and the ion source temperature were set at 240 oC and 250 oC
respectively. Injector temperature was set at 280 oC. The helium gas carrier was maintained at
a linear flow rate of 1 mL/min and the injection volume was 0.1 microliter. An automated
mass spectra library was used for identifying the compounds (NIST, 1990). An internal
standard method was used to quantify the analyzed compounds, taking into consideration the
respective response factors.
Results and Discussions
The urinary dicarboxylic acids profile of Wistar rats (Fig. 2), after oral administration of
ozonized triolein, showed an increment in the concentration of heptanedioic and nonanedioic
acids (Table I) if compared with his respective control. As it could be expected, there were no
appreciated increments of any other dicarboxylic acids. These dicarboxylic acids are formed
taking into consideration the way I of the proposed mechanism (Fig. 1).
Figure 2. Urinary dicarboxylic acids profile of Wistar rats orally treated with ozonized triolein.
Heptanedioic acid (ADC 7), nonanedioic acid (ADC 9) and heptadecanoic acid (AG 17).
By other hand, the urinary dicarboxylic acids profile (Fig. 3) of Wistar rats, after oral
administration of ozonized trilinolein was very similar to that obtained when ozonized
sunflower oil was administered (Jardines et al., 1998). All the dicarboxylic acids were
incremented. A possible explanation of this fact can is given taking into consideration the way
II of the mechanism presented in Fig. 1.
Table I. Rats urinary dicarboxylic acids concentration after oral administration of ozonized
unsaturated compounds
Dicarboxylic acid
Heptanedioic
(Pimelic acid)
Octanedioic
(Suberic acid)
Octenedioic
Nonanedioic
(Azelaic acid)
Decenedioic
Dodecenedioic
Ozonized Triolein
(N=6)
32.6 ± 1
Ozonized Trilinolein
(N=6)
23.4 ± 0.9
Ozonized Sunflower Oil
(N=6)
9.6 ± 0.8
-
0.27 ± 0.05
0.22 ± 0.02
5±1
1.9 ± 0.1
79 ± 4
1.6 ± 0.2
69 ± 4
-
0.040 ± 0.007
0.020 ± 0.005
0.047 ± 0.005
0.043 ± 0.005
Legend: - The concentration is expressed as micrograms of dicarboxylic acid by milligrams
of creatinine.
- The ozonized sunflower oil data are taken from Jardines et al. 1998.
ADC 9
ADC
10:1
NI
NI
ADC 7
16
ADC
12:1
18 t (min.)
AG 17
ADC
8:1
10
20
t (min.)
Figure 3. . Urinary dicarboxylic acids profile of Wistar rats orally treated with ozonized trilinolein.
NI- No identified, heptanedioic acid (ADC 7), octenedioic acid (ADC 8:1), nonanedioic acid (ADC 9),
decenedioic acid (ADC 10:1), dodecenedioic acid (ADC 12:1), heptadecanoic acid (AG 17).
Dicarboxylic acid concentrations obtained in this study were compared with those obtained
when ozonated sunflower oil was administered (Table I). Whit ozonized triglycerides a higher
increment in the urinary dicarboxylic acid concentration was observed. The triglycerides
model compounds (Triolein and trilinolein) and the sunflower oil studied were ozonized in
similar conditions. Nevertheless, the concentrations of some urinary dicarboxylic acids are
significantly different from one substance to another.
The differences could be explained taking into consideration the next facts:
1. The differences in the unsaturated fatty acids composition of triglycerides and the
Sunflower oil. The triolein it is only formed by oleic acid, this fatty acid has one
unsaturation (C9 - C10) and the ozone only react by this position. After the ozone - triolein
reaction the nonanedioic acid is produced and later in the rat organism, could be βoxidized to produce heptanedioic acid, as it is presented en Fig. 1. Nevertheless the
trilinolein has two double bonds in its structure and there are two possibilities for ozone
attack and a higher variety of dicarboxylic acids could be obtained.
The sunflower oil is formed by triglycerides with a 60-65 % of linoleic acid and 20-30 %
of oleic acid in its composition. Although the linoleic acid react with ozone 1.6 times
faster than oleic acid, both compounds react quickly with ozone (k= 10 5 - 10 6 L mol-1 s-1)
at the concentration range they are in the Sunflower oil (Giamalva et al., 1986).
2. The presence of natural antioxidants in vegetable oils that react with ozone, which
although in a small concentration, reduce the ozone - unsaturated fatty acid in the same
proportion. These natural antioxidants are not present in the unsaturated triglycerides used
in this work.
3. The unsaturated fatty acids distribution in the sunflower oil triglycerides. There are some
saturated fatty acids forming part of this triglycerides and it affect in some extension the
ozone - unsaturated fatty acid reaction. It means that there is a different grade of
unsaturated fatty acid accessibility if we ozonized single unsaturated triglycerides or
mixture triglycerides containing saturated and unsaturated triglycerides.
Conclusions
The study of the urinary dicarboxylic acid profile of Wistar rats, previously orally treated with
ozonized unsaturated triglycerides has demonstrated the origin of the increment of these acids
when ozonated sunflower oil was orally administered. The results obtained confirm the
proposed mechanism for ozonized sunflower oil metabolism in rats. The differences observed
were explained taking into account chemical considerations between ozonized sunflower oil
and the unsaturated triglycerides used as models. The urinary dicarboxylic acid profile
depends on the composition of ozonized substance orally administered.
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