Peculiarities of methemoglobin recovery system in erythrocytes of sterlet under nitrite intoxication

Abstract

The content of methemoglobin and the functional status of its recovery system in erythrocytes have been studied for the sterlet Acipenser ruthenus L. exposed to a sodium nitrite concentration ranging from 7.25 to 217.5 mmol/L. The functional features of methemoglobin reductase system form the basis of the specific accumulation of methemoglobin. High concentrations of nitrite in plasma inhibit hemo-containing enzymes (methemoglobin reductase and catalase) that normally prevent the excessive accumulation of methemoglobin. Under the conditions of nitrite intoxication, the leading role in the functioning of the methemoglobin reduction system belongs to its nonenzymatic components (reduced glutathione and ascorbate).

Full text

ISSN 19950829, Inland Water Biology, 2015, Vol. 8, No. 2, pp. 195–199. © Pleiades Publishing, Ltd., 2015.
Original Russian Text © L.V. Khuda, O.I. Khudyi, M.M. Marchenko, 2015, published in Biologiya Vnutrennikh Vod, 2015, No. 2, pp. 99–104.
195
INTRODUCTION
Sterlet is considered to a commercial species of a
high value; it is reared in freshwater ponds because of
its conservation status and nutrition value. One of the
largest native sterlet populations in Ukraine inhabits
the watershed area of the upper reaches of Dniester
River; this demands artificial reproduction and fry
rearing followed by the release of youngsters into nat
ural water bodies. Regard must be paid to stateofthe
art intensive aquaculture methods, particularly using
water recycling facilities (WRF). The recirculation of
water in such plants provides highquality, rapid, and
stable production accompanied by low risks of pan
demic disease; an independence of the production
cycle on natural and climatic conditions; and the total
control of the major water parameters that affect fish
productivity.
Nitrogen derivates demand constant control and
specific attention as one of the major waterquality
factors when using such technology. The entrance of
nitrogen compounds into the aquatic environment is
preconditioned by the metabolism of the aquaculture
species, since ammonia is the main derivate of the pro
tein metabolism in fish. Alongside that, the intensive
rearing of fish in WRF demands fish food of high pro
tein content; it becomes an additional source of nitro
gen in the water.
The ammonia is neutralized by its transformation
to nitrates; this process is performed by nitrification
bacteria at the WRF filter. The high population density
of the fish may lead to the critical concentration of
nitrites in the water and the intoxication of the fish
even at an insignificant lowering of dissolved oxygen in
the rearing tank [16, 18].
The active formation of methemoglobin (MtHb)
and the development of the hemic hypoxia are two of
the most pronounced toxic effects of nitrites. The
methemoglobin content varies in fish in a wide range
due to the effective functioning of the multicompo
nent system of its reactivation [7]. The recovery of
methemoglobin in the erythrocytes is performed by
two enzyme systems; one is linked to glycolysis
(the enzyme NADHmethemoglobin reductase) and
the other to the pentose phosphate pathway (the
enzyme NADHmethemoglobin reductase). In phys
iologically normal conditions, 70–90% of methemo
globin transform into hemoglobin by means
of NADHdependent methemoglobin reductase
(NADHcytochrome
b
5
reductase, EC 1.6.2.2.); this
enzyme is a specific electron transmitter from NADH
via cytochrome
b
5
to MtHb. The NADÐHmethemo
globin reductase is a reserve enzyme system for trans
forming the methemoglobin into hemoglobin; this
enzyme is more physiologically passive and is acti
vated by the exogenous acceptors of the electrons, for
example, by methylene blue or riboflavin. In addition
to the enzymatic pathways of methemoglobin reduc
tion, there is also the possibility of its direct reduction
by the endogenous lowmolecular compounds,
reduced glutathione, and ascorbic acid [6, 15, 17].
Since the erythrocytes provide the media for the tight
interactions between the active oxygen metabolism
and the functioning of the system of methemoglobin
reductase, it is rational to study catalase as well; it is
the hemcontaining enzyme of the antioxidant sys
Peculiarities of Methemoglobin Recovery System in Erythrocytes
of Sterlet under Nitrite Intoxication
L. V. Khuda, O. I. Khudyi, and M. M. Marchenko
Fedkovych National University, Chernivtsi, Ukraine
email: lidia_khuda@email.ua
Received September 29, 2013
Abstract
—The content of methemoglobin and the functional status of its recovery system in erythrocytes
have been studied for the sterlet
Acipenser ruthenus
L. exposed to a sodium nitrite concentration ranging from
7.25 to 217.5 mmol/L. The functional features of methemoglobin reductase system form the basis of the spe
cific accumulation of methemoglobin. High concentrations of nitrite in plasma inhibit hemocontaining
enzymes (methemoglobin reductase and catalase) that normally prevent the excessive accumulation of meth
emoglobin. Under the conditions of nitrite intoxication, the leading role in the functioning of the methemo
globin reduction system belongs to its nonenzymatic components (reduced glutathione and ascorbate).
Keywords
: nitrite, methemoglobin, erythrocytes, sterlet
Acipenser ruthenus
L.
DOI:
10.1134/S199508291502008X
AQUATIC
TOXICOLOGY

196
INLAND WATER BIOLOGY Vol. 8 No. 2 2015
KHUDA et al.
tem, which physiologically prevents hemoglobin from
extra oxidation.
The study aims to assess the effect of nitrite intoxi
cation on the content of methemoglobin and on the
functioning of its reduction system in erythrocytes of
sterlet
Acipenser ruthenus
L.
MATERIALS AND METHODS
The isolated erythrocytes of youngoftheyear of
sterlet (average body weight of 350 g) has been used to
model the effect of nitrites on the functioning of the
recovery system of methemoglobin. The blood is sam
pled in the dorsal aorta and heparin is added immedi
ately to prevent coagulation. The erythrocytes are iso
lated by centrifuge at 500 g and are washed from the
plasma remains in a Ringer solution three times after
centrifuging. The erythrocytes are divided by the dilu
tion into six groups, one was control, and the other five
are experimental. All erythrocytes are incubated at
20
°
С
in the Ringer solution for 30 min; the concentra
tion of
NaNO
2
, mmol/L, is set for the experimental
groups as 7.25 (I group), 14.5 (II), 72.5 (III),
145.0 (IV), and 217.5 (V).
The semilethal concentration of nitrite ions in the
water is considered to be 1.45 mmol/L for many fresh
water fish species [10, 18]. Taking into account the
tenfold accumulation of nitrite ions in blood plasma of
fish [13], the concentration of this compound in the
erythrocyte incubation media is increased accordingly.
The content of methemoglobin (% of total hemo
globin) is analyzed by spectrophotometry using the
acetone cyanhydride method [2]. The relative activity
of methemoglobin reductase (
µ
mol/min per 1 mg of
hemoglobin) is assessed using the recovery rate of
methemoglobin in the presence of NADH [2]. The
relative activity of catalase (mmol/min. Per 1 mg of
protein) is assessed using the reaction of utilization of
hydrogen peroxide with ammonia molybdate [5]. The
concentration of the reduced glutathione (mmol/g of
protein) are assessed using its reaction with 2,2'Dini
tro5,5'dithiodibenzoic acid [5], reduced ascorbic
acid (
µ
mol/mg of hemoglobin) under the difference
between all the derivates of ascorbate and sum of dehy
droascorbic acid and diketogulonic acid [2]. The con
tent of total protein (mg/mL) is assessed by Lowry
method; that of hemoglobin (Hb, mg/mL) is assessed
by the hemoglobin cyanide method [4].
RESULTS
A high concentration of methemoglobin was
observed in the erythrocytes of all experimental
groups. However, the most pronounced accumulation
of MtHb was observed in the erythrocytes that were
incubated at the semilethal nitrite concentration, or
similar ones (experimental groups II and III). In these
groups, MtHb concentration was about 50% of total
hemoglobin concentration (Fig. 1a). The use of higher
nitrite concentrations (145 and 217.5 mmol/L) was
accompanied by the decrease of methemoglobin con
centration; however, these values exceeded the control
range 2.6 and 2.8 times, respectively.
When studying methemoglobin reductase activity,
it was found that the incubation of the erythrocytes
with the nitrites at the concentrations lower than
145 mmol/L did not result in significant changes of
this parameter, and the methemoglobin reductase
activity was similar to control range (Fig. 1b). In
group IV, the methemoglobin reductase activity
decreased 1.4 times compared to control.
The activity of catalase, which is another hemcar
rying enzyme of erythrocytes, was low at all the studied
nitrite concentrations, and the minimal values have
been observed for the erythrocytes of groups I and III
(Fig. 1c).
The components of the nonenzyme pathway of the
recovery system of methemoglobin comprise glu
tathione and ascorbic acid. Both parameters
responded in accordance to the nitrite concentration
and differed significantly from the range obtained for
the control group at all the studied nitrite concentra
tions. The decrease in the reduced glutathione con
centration in the erythrocytes of groups III (by 42%)
and IV (by 58%) indicates its active role in the recovery
of methemoglobin at a
NaNO
2
presence of 72.5 and
145 mmol/L (Fig. 1d). The incubation of sterlet erythro
cytes at lower nitrite concentrations (groups I and II) was
accompanied by an increase of GSH when compared
to the control group.
The increase in nitrite concentration in the incuba
tion media has been accompanied by the decrease in
reduced ascorbate concentration in erythrocytes
(Fig. 1d). A significant decrease in this parameter has
been observed even for group I (
~1.5
times when com
pared to the control); the minimal concentration of
ascorbic acid has been registered at
NaNO
2
concen
tration of 217.5 mmol/L.
DISCUSSION
Fish hemoglobin is oxidized easily, so the concen
tration of MtHb may vary in a wide range even at phys
iological conditions. The absence of obvious intoxica
tion in that case is explained by the effective function
ing of the multicomponent methemoglobin reductase
system [1, 7].
The functional peculiarities of the system of MtHb
recovery may form the basis in the accumulation of
methemoglobin in erythrocytes of sterlet at nitrite
intoxication. NADHcytochrome
b
5
reductase
(NADHmethemoglobin reductase or diaphorase1),
which uses NADH synthesized by the glyceraldehyde
phosphate dehydrogenase reaction of glycolysis, is a
major component promoting the normal recovery of
MtHb into hemoglobin in all vertebrates, including
fish [6, 17]. The electron transmission from NADH
via cytochrome
b
5
to the molecule of oxidized hemo

INLAND WATER BIOLOGY Vol. 8 No. 2 2015
PECULIARITIES OF METHEMOGLOBIN RECOVERY SYSTEM 197
globin is accompanied by the transformation of the
hem trivalent ferric to the bivalent form.
The results of our study, however, do not indicate
the active response reaction of this sterlet erythrocyte
enzyme upon an increase in MtHb; i.e., the activity of
methemoglobin reductase in four experimental groups
did not differ from control, and it was inhibited only at
a
NaNO
2
concentration of 145 mmol/L. A decrease in
the methemoglobin reductase activity at all studied
nitrite concentrations has been registered earlier in the
erythrocytes of crucian carp [8].
The interaction of nitrites with the ion of ferric of
the hemoglobin hem allows us to suppose the possibil
ity of such reactions with other hemcarrying proteins,
particularly, cytochrome
b
5
as a component of methe
moglobin reductase [14]. In addition, the catalase
60
20
40
%
(a)
*
*
*
*
*
0.6
1.2
mmol/min per 1 mg of protein
(c)
*
*
*
*
*
0.15
0.30
µ
mol per 1 mg of Hb
(e)
*
*
*
*
*
0.45
К 7.25 14.5 72.5 145.0 217.5
mmol/L
mmol/min per 1 mg of protein
(d)
*
*
*
*
0.4
К 7.25 14.5 72.5 145.0 217.5
mmol/L
0.2
*
µ
mol/min per 1 mg of Hb
(b)
2
1
*
Fig. 1.
Methemoglobin (a), relative activity of methemoglobin reductase (b) and catalase (c), and reduced glutathione (d) and
ascorbate (e) in the erythrocytes of sterlet exposed to different concentrations of NaNO
2
, mmol/L: (K) control; *statistically sig
nificant differences in regard to the control.

198
INLAND WATER BIOLOGY Vol. 8 No. 2 2015
KHUDA et al.
activity of sterlet erythrocytes may evidence the inhib
iting effect of nitrite ions on methemoglobin reductase
complex due to the changes in oxidizing the ferric ion
(in hem). Catalase comprises for identical subunits,
each of which carries a prosthetic hem group that
include the ferric ion. The nitrite anions may react
with the hem ferric in the activity center of catalase
and inhibit its activity at a concentration of
10
–3
and
higher [3].
The functioning of the methemoglobin recovery
system in the erythrocytes is tightly linked to the func
tioning of the antioxidant system. On the one hand,
the accumulation of MtHb causes the release of a
superoxide anion that, in turn, promotes the synthesis
of
H
2
O
2
, which can reduce and release the hydroxyl
radical
ОН
•
. On the other hand, the ions of
Fe
2+
,
which are released during the abundant synthesis of
MtHb, promote the initiation of freeradical processes
[11]. The effect of
NaNO
2
at studied concentrations
leads to the intensification of the oxidizing processes
in fish erythrocytes and the accumulation of derivates
of the oxidized modification of lipids and proteins [9].
High concentrations of nitrites may inhibit the activity
of antioxidant enzymes, particularly, superoxide dis
mutase, peroxidase, and catalase, together with the
direct damage of the protein molecules [3].
Probably the alternative pathways guided by non
enzyme lowmolecular compounds become leaders in
reducing methemoglobin during the experiments per
formed in this study. Ascorbic acid and reduced glu
tathione have the ability to directly reduce MtHb at
nitriteinduced methemoglobinemia.
A high concentration of glutathione GSH is usual
for fish erythrocytes. The GSH/Hb ratio in fish is sig
nificantly higher than in mammals [7]. The redox sys
tem of glutathione (GSHGSSG) serves as a buffer
that prevents the destructive effect of active forms of
oxygen and supports the mechanisms of detoxication.
The sulfhydryl (reduced) form of glutathione reacts
easily to enzyme and nonenzyme oxidation and results
as a disulfide (oxidized) form of glutathione. Being an
effective antioxidant, glutathione plays an exceptional
role in supporting the structural integrity of the eryth
rocytes and protects SH groups of hemoglobin and
other proteins of erythrocytes from the influence of
oxidizing agents. Therefore, the possibility of directly
reducing methemoglobin and antioxidant features of
GSH preconditions its significant role in the supporting
system of the hemoglobin’s structure and functions.
On one hand, our data report on introducing the
reduced glutathione to the response of erythrocytes to
the nitrite intoxication at relatively high concentra
tions of
NaNO
2
(72.5 and 145 mmol/L); this is sup
ported by a decrease in glutathione concentration. On
the other hand, a high concentration of GSH in eryth
rocytes of groups I and II may promote the effective
functioning of ascorbate, i.e., another redox system.
The reduction of dehydroascorbic acid into ascorbic
acid takes less time in the presence of sulfhydrylcar
rying complexes such as cysteine and glutathione [6].
Ascorbate has a direct reducing effect on methe
moglobin in erythrocytes, as does GSH. Ascorbate is
used widely in the therapy of nitriteinduced methe
moglobinemia in humans [6]. Contradictory data have
been found in the literature in regards to the activity of
Lgulono
γ
lactone oxidase (the last enzyme in the
biosynthesis of ascorbic acid from glucose) in Aci
penseridae, including sterlet particularly. However,
despite the possibility of synthesis of ascorbate by Aci
penseridae, many authors agree on the necessity of
adding extra vitamin C into the fish food [12, 19].
Our results evidenced the rapid involvement of
ascorbate into the reaction of erythrocytes in the pres
ence of nitrite. A significant decrease in the concen
tration of reduced ascorbate was observed upon an
increase of
NaNO
2
in the incubation media. This was
probably linked to the transformation of reduced
ascorbic acid into dehydroascorbic acid after it was
used as the reducing agent and antioxidant.
CONCLUSIONS
The functional features of methemoglobin reduc
tase system form the basis of the specific accumulation
of methemoglobin in erythrocytes of sterlet. High
concentrations of nitrite in plasma inhibit hemocon
taining enzymes (methemoglobin reductase and cata
lase), which normally prevent the excessive accumula
tion of methemoglobin. Under the conditions of
nitrite intoxication, the leading role in the functioning
of the methemoglobin reduction system belongs to its
nonenzymatic components (reduced glutathione and
ascorbate).
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