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V.M. Korobko et al./Nitric Oxide 42 (2014) 62–69
presence of cytochrome c. For this purpose we studied the
following problems:
on the skin surface with fibers of the probe. The movement of eryth-
rocytes causes a Doppler shift, which in turn is detected by the laser
light and analyzed by the LAKK-02. This is then computed and dis-
played as the blood flow velocity. The detected laser signal correlates
with the number of moving erythrocytes in tissue, blood flow ve-
locity for calculation microcirculation parameters, using such
arbitrary (relative) units as perfusion units (perf. un.). The rate of
microcirculation (the microcirculation level), the regulatory
activity of its components and the degree of shunt paths partici-
pation with an allowance for the frequency range intervals of the
blood flow oscillations in the rats’ microvessels were investigated
Heart rate variability (HRV) was studied with the help of the
“Neurosoft” (Ivanovo, Russia) system with their own adaptation of
ECG registration and results management [23]. Time parameters in-
cluded parameters such as Mo, which is the most frequent mode
of RR-cardiointerval values (RR intervals were registered for 5
minutes within 0.001 s in the morning hours in rats at rest), AMo
amplitude of modal value of RR interval set, Standard Deviation of
Normal-to-Normal (SDNN) RR intervals, tension index (TI).
Statistical treatment of the results was performed with the
Statistica 6.0 program. All heart rate variability data were given with
at least two significant digits, except p-values (one significant digits)
and were presented as mean relative standard deviation (RSD, %).
Two-tailed tests were used. The lower level of statistical signifi-
cance was set to p = 0.05. All means were accompanied by their 95%
confidence intervals.
the estimation nitrosyl complexes of cytochrome c with
nitromedicine and the role of sodium ascorbate in the forma-
tion of nitric oxide “depot” from complexes by UV-visible spectra;
•
the study of burn wound regeneration in the experiments in vivo
by rats;
•
the detection of the increase of total [NOx] concentration in blood
plasma in vivo under treatment of nitromedicine-cytochrome
c-complexes;
•
the estimation of useful side pharmacological effects such as
vasodilatation.
•
2. Experimental section
Materials and reagents. Cytochrome
c (from heart
bovine) (≥95%, lot STBB7839V, Fluka (USA), Sigma-Aldrich),
tris(hidroxymethyl)aminomethane (>99.2%, lot 108387, Merck), sol-
vents (analytical grade), 5-nitrofural (>99.8%), metronidazole (>99.8%),
sodium nitrite (>99.9%), 5-nitroxolin (>99.8%), ascorbic acid (>99.9%),
phosphoric, succinic acid were purchased from Sigma-Aldrich and
used without further purification.
NO were prepared by the addition of 2 M H2SO4 to solid NaNO2
in a Kipps apparatus. The NO gas was passed through four NaOH
(20%) traps (to remove NO2), and then through a solid CO2 trap. The
gas was collected in a buffer solution that had undergone four
vacuum/N2 deoxygenation cycles. The concentration of the NO so-
lution varied from 1.2 to 2 mM. The NO2− concentration was generally
approximately 300 μM.
3. Results and discussion
3.1. UV-visible study of reaction mixture with nitromedicine
generated NO-containing complexes
Nitrite was assayed by the Griess reaction [18]. One hundred
microliter probes or standards were added to 500 μl of 1% (w/v)
sulfanilamide in 5% (v/v) H3PO4 and 500 μl 0.15 napthylenediamine
hydrochloride. The optical density (A) was measured at 540 nm, and
the assay was calibrated using nitrite standards. Determination of
nitrite and nitrate using the Griess reaction was carried out
according to methods by using Aspergillus nitrate reductase.
Absorption spectra of aqueous solutions (nitromedicine, cyto-
chrome c, organic acids, phosphoric acid and their mixtures) were
recorded by Bio line Specord S-100 (Analytik Jena), with thickness
10 mm quartz cuvette. Spectrophotometer error is 0.002 units trans-
mittance at a concentration of cytochrome c 4.5·10–5 mol/L–1 in
solution, and the relative standard deviation (RSD%) was 0.9%.
Formulation of pharmaceutical composition. Composition of
gel (w, %): sodium hyaluronate – 1.0; hydroxyethylcellulose – 0.1;
sodium ascorbate – 0.1; cytochrome c – 0.05; methylparaben – 0.15;
water up to 100. Sodium nitrite (0.1–1%) was added into gel before
application. Composition of powder (w, %): cytochrome c −0.05;
sodium ascorbate – 0.1; 5-nitrofural (metronidazole, nitroxolin) –
5%; methylparaben – 0.15; starch up to 100. Pharmaceutical com-
position (gel plus sodium nitrite) should be prepared just before use.
Biomedical research. The experiment with nitromedicine con-
taining dosage form was carried out by using twenty Wistar male
rats and ten rats were used as the control group. Two groups of
animals were separated, that were exposed to contact thermal burn
under anesthesia (the burn area is 20% of the body surface) [19].
After thermal injury modeling a pharmaceutical composition which
was a complex of cytochrome c and sodium nitrite was applied to
the wound surface of the animals in the main group (n = 20) for 10
days. The animals in the control group (n = 10) were treated with
5-nitrofural-containing ointment.
Sodium ascorbate was used for a spectrum control of cyt c re-
ducing form by using absorption of α- (550 nm), β- (520 nm) and
γ- (415 nm) bands. In this case at pH 7.1, the initial oxidizing form
of cytochrome c (cyt c3+), which bands at λmax 410 nm converts to
reducing form of cyt c2+ in full (Fig. 1a). Fig. 1b shows the changes
of UV-visible spectrum of aqueous solution of cyt c following the
addition of gaseous NO during 15 minutes. The shift of γ-band at
410 nm to 415 nm characterized reducing form was observed. More-
over, new intensive band at 358 nm, two weak bands – 520 nm (β)
and 550 nm (α) typical for cyt c2+ –appeared.
The same spectral effect was estimated when cyt c was inter-
acted with nitrite at pH 3 in the work [24]. At this pH the UV as
well as visible spectrum of cyt c was changed by nitrite, even in the
presence of hydrogen peroxide, probably via the formation of heme
iron-nitric oxide complex. This spectrum argument of a heme nitric
oxide complex of cyt c had been reported previously by Orii and
Shimada (1978) and Butt and Keilin (1962). Due to this change, the
peroxidase activity of cyt c was lost.
The similar change of the heme structure of cyt c was shown by
absorption spectra in the UV visible range in alternative system, con-
taining a mixture of cyt c, sodium nitrite and sodium ascorbate in
acid medium, able to generate nitric oxide (Fig. 2a). The UV-Vis spectra
of the reaction mixtures with succinic acid are represented in Fig. 2a.
We can see the shift of γ-band from 410 nm to 415 nm, the appear-
ance of new intensive band at 358 nm, weak α- and β-bands such
as in the spectrum of cyt c3+ under gaseous NO action (Figs. 1,2). The
significant differences of cyt c spectra of reaction mixtures, con-
taining sodium ascorbate and nitrite at pH 5.0, had the reversible
shift of γ-band from 415 nm to 410 nm in a time – 30 minutes (Fig. 2b).
At the same time, a new band at 358 nm was changed in a slight
variation of time, but α- and β-bands disappeared.
The microcirculation was assessed quantitatively using the
LAKK-02 (LASMA, Russia). This device transmits continuous wave
laser light (30 mW, 890 nm) and white light (20 W, 500–900 nm)
to skin tissue near the wound, where it is scattered and collected
The type and character of spectra depended on pH medium
and biogenic acid nature. Analysis of spectra data of the reaction
Korobko
