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



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).
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.
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
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
reductase, EC; this
enzyme is a specific electron transmitter from NADH
via cytochrome
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
Received September 29, 2013
—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).
: nitrite, methemoglobin, erythrocytes, sterlet
Acipenser ruthenus
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
Acipenser ruthenus
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
in the Ringer solution for 30 min; the concentra
tion of
, 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 (
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 (
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].
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
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 (
times when com
pared to the control); the minimal concentration of
ascorbic acid has been registered at
tration of 217.5 mmol/L.
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
(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
to the molecule of oxidized hemo
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
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
as a component of methe
moglobin reductase [14]. In addition, the catalase
mmol/min per 1 mg of protein
mol per 1 mg of Hb
К 7.25 14.5 72.5 145.0 217.5
mmol/min per 1 mg of protein
К 7.25 14.5 72.5 145.0 217.5
mol/min per 1 mg of Hb
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
, mmol/L: (K) control; *statistically sig
nificant differences in regard to the control.
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
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
, which can reduce and release the hydroxyl
. On the other hand, the ions of
which are released during the abundant synthesis of
MtHb, promote the initiation of freeradical processes
[11]. The effect of
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
(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
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
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.
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
1. Andreeva, A.Yu. and Soldatov, A.A., The sensitivity of
nucleated red cells of
Scorpena porcus
L. to different
nitrite concentrations (experiments
in vitro
), in
III Mezhdunar. ikhtiol. nauch.prakt. konf.
III Int. Ichthyol. Sci.Pract. Conf.), Dnepropetrovsk,
2010, pp. 10–12.
2. Goryachkovskii, A.M.,
Klinicheskaya biokhimiya v lab
oratornoi diagnostike
(Clinical Biochemistry in the
Laboratory Diagnosis), Odessa: Ekologiya, 2005.
3. Maevska, O.M., Boiko, M.M., and Velikii, M.M.,
Influence of sodium nitrite on catalase activity,
Biokhim. Zh.
, 2004, vol. 76, no. 5, pp. 140–143.
Metodicheskie ukazaniya po provedeniyu gematolog
icheskogo obsledovaniya ryb. no. 1342/1487
ological Guidelines for Hematologic Examination of
Fish, no. 1342/1487), Moscow: Minsel’khozprod
RF, 1999.
Metody otsenki oksidativnogo statusa
(Methods of
Assessment of Oxidative Status), Voronezh: Izd.Poli
graf. Tsentr Voronezh. Gos. Univ., 2009.
6. Prodanchuk, G.N. and Balan, G.M., Toxic methemo
globinemia: mechanisms of formation and ways to opti
mize treatment,
Sovrem. Problemy Toksikol.
, 2007,
no. 1, pp. 37–45.
7. Soldatov, A.A., Structure, polymorphism, and resis
tance to oxidation of fish hemoglobins,
Zh. Evol.
Biokhim. Fiziol.
, 2002, vol. 38, no. 4, pp. 305–308.
8. Khuda, L.V., Marchenko, M.M., Khachman, Ya.Yu.,
and Khudii, O.I., Effect of nitric intoxication on the
methemoglobin recovery system in erythrocytes of sil
ver carp,
Biol. Sist.
, 2012, vol. 4, no. 4, pp. 393–396.
9. Khuda, L.V., Marchenko, M.M., and Khudii, O.I.,
Intensity of oxidative processes in carp erythrocytes
under nitric intoxication,
Biol. Sist.
, 2013, vol. 5, no. 1,
pp. 16–21.
10. Alexander, J., Benford, D., and Cookburn, A., Nitrite
as undesirable substances in animal feed,
2009, vol. 1017, pp. 1–47.
11. Cooper, Ch., Peroxynitrite reacts with methemoglobin
to generate globinbound free radical species,
Adv. Exp.
Med. Biol.
, 1999, vol. 454, pp. 195–202.
12. Gy, Papp Zs., Jeney, Zs., and Jeney, G., Comparative
studies on the effect of European catfish (
Silurus glanis
and sturgeon hybrid (
Acipenser ruthenus
J. Appl. Ichthyol.
, 1995, vol. 11, pp. 372–374.
13. Kroupova, H., Machava, J., and Piackova, V., Nitrite
intoxication of common carp (
Cyprinus carpio
L.) at
different water temperatures,
Acta Vet. Brno
, 2006,
vol. 75, pp. 561–569.
14. Moraes, G., Avilez, I.M., and Altran, A.E., Biochemi
cal effects of environmental nitrite in matrinxa (
), in
Aquatic Toxicology: Mechanisms and Con
sequences. International Congress on the Biology of Fish.
Symposium Proceedings,
Vancouver, 2002, pp. 15–26.
15. Padayatty, S.J., Katz, A., Wang, Y., et al., Vitamin C as
an antioxidant: evaluation of its role in disease preven
J. Amer. College Nutrit.
, 2003, vol. 22, no. 1,
pp. 18–35.
16. Raja, I.A. and Sapkal, H.P., Blood and electrolyte
responses in
Clarias batrachus
exposed to nitrogen pol
Biosci. Biotech. Res. Comm.
, 2011, vol. 4, no. 2,
pp. 219–222.
17. Saleh, M.C. and McConkey, S., NADHdependent
cytochrome b
reductase and NADPH methemoglobin
reductase activity in the erythrocytes of
Fish Physiol. Biochem.
, 2012, vol. 38, no. 6,
pp. 1807–1813.
18. Svobodova, Z., Machova, J., and Poleszczuk, G.,
Nitrite poisoning of fish in aquaculture facilities with
waterrecirculating system,
Acta Vet. Brno
, 2000,
vol. 74, pp. 129–137.
19. Verlhac, V. and Gabaudan, J.,
The Effect of Vitamin C on
Fish Health. Roche Technical Bulletin
, Basel: Hoff
mannLa Roche Ltd., 1997.
Translated by D. Martynova
ResearchGate has not been able to resolve any citations for this publication.
Full-text available
Three cases of severely compromised fish health and death in newly commissioned aquaculture facilities with water-recirculating systems are described. The cause of the damage and death was increased concentrations of water-borne nitrites and the subsequent methaemoglobinemia. The aim of the study was to better understand the aetiology of these cases of poisoning to help prevent them, and to examine effects of some water quality parameters on nitrite toxicity. The increased NO 2- concentrations in water were caused by impaired functionality of biological filters in the second stage of nitrification, i.e. the conversion of NO 2- to NO 3-. Chloride concentrations in water were considered the main factor influencing NO 2- toxicity in all of the cases described. In the case of death of catfish and tench, the Cl -to N-NO 2- weight ratios were in the range of 13-28 and 11-19, respectively. In the case of tilapia health impairment without symptoms of toxicity, the ratios were between 50 and 150. In the water tank inflow, the Cl -to N-NO 2- weight ratios were between 2000 and 10000. Blood methaemoglobin levels of catfish and tench (severe symptoms of poisoning) and of tilapia (no signs of impairment, only brownish discolouration of gills) were over 80% and 21%, respectively). In order to minimize risks in culture of fish in water-recirculating systems, it is necessary to choose a proper stock of fish and a proper feeding ratio, not to treat the fish with antibiotics in the form of baths, to check meticulously the quality of water. In case of increasing concentration of nitrites, to administer sodium chloride to get the chloride concentration increased at least to 100 mg·l -1 Cl -. Better operation of a biological filter can be speeded up by inoculation with activated sludge.
Full-text available
Common carp (Cyprinus carpio L.) were exposed to nitrite (1.45 mmol·l -1 NO 2-) for 48 hours at 14°C and 20°C, in order to investigate the mechanism of nitrite poisoning at these water temperatures. The effect of nitrite exposure on fish was assessed on selected haematological and biochemical indicators of the blood. Moreover, nitrite accumulation in the blood, liver and muscle was measured. Nitrite exposure produced high levels of methaemoglobin (88.2 ± 3.3% and 92.9 ± 6.1%) at both water temperatures compared with controls (0.3 ± 0.6% and 2.6 ± 3.0%). High fish mortality occurred in experimental groups (30% and 51%) compared with controls (0%). Nitrite exposure also resulted in an accumulation of nitrite in the fish body. The highest nitrite levels developed in the blood plasma, followed by the liver and muscle, respectively. Carp concentrated nitrite in the blood plasma and tissues to markedly higher levels at higher temperature (20°C). The plasma nitrite concentrations (10.5 ± 1.9 mmol·l -1) were in this case more than 7 times higher than the environmental one. At lower temperature (14°C), plasma nitrite concentration reached 5.0 ± 1.5 mmol·l -1. In either event, plasma K + levels increased and Cl - levels and osmolality remained unchanged. Plasma Na + levels slightly decreased at the higher temperature. Nitrite-exposed fish showed lower haematocrit values (PCV) at both experimental temperatures compared with controls. At 20°C, the blood haematocrit decrease (0.20 ± 0.02 l·l -1) was accompanied by a low erythrocyte count (1.05 ± 0.12·10 12 l -1) and by a low haemoglobin level (51 ± 11 g·l -1). At the lower temperature (14°C), the haematocrit decrease (0.25 ± 0.02 l·l -1) was caused by a low mean corpuscular volume (167 ± 27 fl). No significant changes were observed in the mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), or selected erythrocyte dimensions (major axis, minor axis and aspect ratio).
Full-text available
Vitamin C in humans must be ingested for survival. Vitamin C is an electron donor, and this property accounts for all its known functions. As an electron donor, vitamin C is a potent water-soluble antioxidant in humans. Antioxidant effects of vitamin C have been demonstrated in many experiments in vitro. Human diseases such as atherosclerosis and cancer might occur in part from oxidant damage to tissues. Oxidation of lipids, proteins and DNA results in specific oxidation products that can be measured in the laboratory. While these biomarkers of oxidation have been measured in humans, such assays have not yet been validated or standardized, and the relationship of oxidant markers to human disease conditions is not clear. Epidemiological studies show that diets high in fruits and vegetables are associated with lower risk of cardiovascular disease, stroke and cancer, and with increased longevity. Whether these protective effects are directly attributable to vitamin C is not known. Intervention studies with vitamin C have shown no change in markers of oxidation or clinical benefit. Dose concentration studies of vitamin C in healthy people showed a sigmoidal relationship between oral dose and plasma and tissue vitamin C concentrations. Hence, optimal dosing is critical to intervention studies using vitamin C. Ideally, future studies of antioxidant actions of vitamin C should target selected patient groups. These groups should be known to have increased oxidative damage as assessed by a reliable biomarker or should have high morbidity and mortality due to diseases thought to be caused or exacerbated by oxidant damage.
Methemoglobin is oxidized hemoglobin that cannot bind to or dissociate from oxygen. In fish, it is most commonly caused by exposure to excess nitrites and can lead to abnormal swimming, buoyancy, or death. The methemoglobin concentration in mammals is determined by the balance of oxidizing agents versus reducing enzymes in erythrocytes. The objective of our studies was to characterize the enzymes that reduce methemoglobin in fish erythrocytes. Whole blood was collected from healthy rainbow trout. Methemoglobin was induced in vitro by NaNO(2) exposure. Methemoglobin reduction in controls was compared to reduction in samples with added NADH, NADPH, or NADPH and methylene blue. Rainbow trout whole blood was also fractionated into cytosol, microsomal, and mitochondria/plasma membranes/nuclei fractions. The fractions were compared for NADH-dependent cytochrome b5 reductase (CB5R) activity and for nitrite induction of methemoglobin. The CB5R activity in rainbow trout erythrocytes was compared to the CB5R activity in equine, feline, and canine erythrocytes. Rainbow trout erythrocytes had significant NADPH methemoglobin reductase activity in the presence of methylene blue (P < 0.001). The CB5R activity was greatest (P < 0.001) in the plasma membrane/mitochondria/nuclei fraction. The CB5R activity in rainbow trout erythrocytes was not significantly different than canine or equine activity but was significantly lower than feline CB5R activity (P < 0.0001). Methemoglobin in rainbow trout erythrocytes can be reduced by CB5R or NADPH-dependent methemoglobin reductase. Unlike mammalian anuclear erythrocytes, which are dependent on soluble CB5R, the nucleated RBCs of rainbow trout use membrane-bound CB5R to reduce methemoglobin.
Reactive oxygen species been implicated in oxidative stress and vascular injury. These include the free radicals Superoxide (O2⋅)’ hydroxyl (OH⋅), peroxyl (LO2⋅) and alkoxyl (LO⋅) and the non-radicals hydrogen peroxide (H2O2), lipid peroxides and singlet oxygen. It was originally thought that the hydroxyl radical (OH⋅) was likely to be the most damaging species (1); however, this free radical is so reactive that it has a diffusion distance of no more than a few nm at most (2). The hydroxyl radical’s non-specific reactivity also makes it less likely that it will react with a vital cellular component. Other less reactive free radicals may be more toxic in that they can mediate damage at some distance from where they are formed.
It was shown in vitro that the activity of catalase Penicillium vitale decreases when treated by sodium nitrite to the greater extent than when affected by dipeptide carnosine (beta-alanyl-L-histidine). At alternating introduction of carnosine and NaNO2 that component affected the catalase activity which was the first to be introduced. The entering of the next metabolite into the medium did not change the enzyme activity. It is shown that carnosine binds with a molecule of catalase. Carnosine may be considered one of natural regulators of catalase activity.
Klinicheskaya biokhimiya v lab oratornoi diagnostike
  • A M Goryachkovskii
Goryachkovskii, A.M., Klinicheskaya biokhimiya v lab oratornoi diagnostike (Clinical Biochemistry in the Laboratory Diagnosis), Odessa: Ekologiya, 2005.