Reprod Dom Anim 37, 27±30 (2002)
Ó 2002 Blackwell Wissenschafts-Verlag, Berlin
ISSN 0936-6768
Placental Release/Retention in Cows and its Relation to Peroxidative
Damage of Macromolecules
M Kankofer
Department of Biochemistry, Faculty of Veterinary Medicine, Agricultural University, Lublin, Poland
Contents
The disturbances in metabolic pathways re¯ected in clinical
symptoms of illnesses may be connected, among others, with
the imbalance between production and neutralization of
reactive oxygen species. One of such illnesses may be the
retention of fetal membranes in cows. The levels of reactive
oxygen species can be measured directly or estimated indirectly
by the determination of enzymatic and non-enzymatic antioxidative defence systems. The determination of parameters
indicating the intensity of peroxidative processes of lipids,
proteins and nucleic acids caused by reactive oxygen species is
also useful. This review examined the available literature
regarding peroxidative processes of lipids, proteins and nucleic
acids caused by reactive oxygen species as well as parameters
indicating its intensity. All information relates the importance
of proper and improper placental release in cows.
Introduction
Reactive oxygen species (ROS) are intermediates which
are produced during metabolism. Their level is controlled by enzymatic and non-enzymatic defence mechanisms which are able to neutralize them by di€erent
types of biochemical reactions (Sies 1993). ROS may
have a positive role such as involvement in killing
bacteria by phagocytic cells (Halliwell 1987).
However, when ROS levels increase in an uncontrolled way, they may exhibit direct and indirect
negative e€ects. The direct negative e€ects of ROS
excess are peroxidative damage to biologically important macromolecules which, in turn, may lead to peroxidative changes in cell membranes, degradation of cell
structures, lysis of cells and damage to the tissues
(Halliwell and Guteridge 1985). Indirect negative e€ects
are connected with peroxidative inactivation of steroidogenic (Takayanagi et al. 1986) and arachidonic acid
cascade enzymes. ROS may cause disturbances in
NADP/NADPH ratios leading to improper function
of some enzymes and alterations in the metabolism
(Golden and Ramdath 1987).
All these alterations on molecular, cell and tissue level
may ®nally be re¯ected in clinical symptoms of di€erent
illnesses. This hypothesis might be based on the determinations of increased ROS or its metabolite levels and/
or decreased antioxidative eciency which leads to
imbalance between the production and neutralization of
ROS. There is also evidence that diet supplementation
with antioxidants decreases the risk of such imbalances
and decreases the risks of some diseases such as
circulatory disturbances (Kleijnen et al. 1989). Examples of diseases, for which the aethiology can be
considered in terms of ROS, are: cancer, artheriosclerosis, Parkinson's disease, neurological diseases, arthritis
U.S. Copyright Clearance Centre Code Statement:
and AIDS (Halliwell 1987). Evidence for a connection
between ROS imbalance and the retention of fetal
membranes in cows has been researched (Miller et al.
1993). The disturbances in steroid hormones and prostaglandins, among others, during the retention of fetal
membranes have been described (Leidl et al. 1980;
Grunert 1983).
The ROS, however, are substances of di€erent chemical characters that can be detected both directly and
indirectly. Direct ROS determination is dicult mainly
because of their short half lives. Electron paramagnetic
resonance spectrometry is one of the possible methods,
but very often its sensitivity is too low for biological
samples. It can be increased by measurements performed
at the temperature of liquid nitrogen (Bartosz 1995). The
spin trap method provides the possibility to determine
radical adducts of ROS with adequate nitrogen compounds such as 5,5 dimethyl-1-pyrol-N-oxide (DMPO).
Monomolar or dimolar chemiluminescence detection is
also useful. ROS may react also with di€erent substances
creating non-radical connections which can be determined by di€erent means, for example high performance
liquid chromatography (HPLC).
Indirect estimation is based on the analysis of defence
mechanisms against ROS. These include the determination of mRNA expression and activity of antioxidative
enzymes such as: glutathione peroxidase (GSH-Px),
glutathione transferase (GSH-Tr), superoxide dismutase
(SOD) and catalase (CAT). The methods for the determination of non-enzymatic antioxidants such as: glutathione, water and lipid-soluble vitamins (vitamin C, A
and E, respectively) are also available, mainly by spectrophotometry and spectro¯uorimetry. Indirect estimation also covers the determination of end products or
intermediates of peroxidative processes of macromolecules such as lipids, proteins and nucleic acids.
The main objective of this review is to describe the
relation between bovine placental release/retention processes and peroxidative damage of lipids, proteins and
nucleic acids. Some possibilities of indirect estimation of
excess ROS that appear during the retention of fetal
membranes in cows are also discussed.
Lipid peroxidation
Lipid peroxidation is a non-enzymatic chain reaction
based on oxidation of mainly unsaturated fatty acids
and is associated with the presence of ROS. It leads to
the creation of lipid peroxides and other intermediates.
These intermediates may in¯uence the properties of cell
membranes and their physiological functions (Yagi
1982; Halliwell and Guteridge 1985).
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28
Enzymatic peroxidative processes of unsaturated fatty
acids, which are catalysed by lipo or cyclo-oxygenases,
lead to the formation of biologically active substances
such as prostaglandins, leukotrienes or thromboxanes
(Kuehl and Egan 1980).
Lipid peroxidation can be caused by hydroxyl,
peroxyl or alloxyl radicals of the substances present in
cells or by xenobiotics. Peroxidation processes consist of
three steps: initiation, propagation and termination
(Bartosz 1995). Initiation involves the detachment of
hydrogen from free or phospholipid-linked unsaturated
fatty acids leading to lipid radical formation (Eichenberger et al. 1982). During the propagation step, the
process expands to involve new molecules. As a result of
these steps the products of lipid peroxidation ± hydroperoxides and conjugated dienes may be created. Termination is connected with the recombination of
radicals that leads to the formation of non-radical
products. The most common are malondialdehyde
(MDA) and 4-hydroxynonenal (Comporti 1989). Their
toxicity is, among others, based on anity to thiol
groups and formation of Schi€ base-type connections
with amino acids (Haberland et al. 1994). These lead to
changes in enzyme activities and reactions with nucleic
acids (Dianzani 1982).
The consequences of lipid peroxidation processes may
be associated not only with the presence of toxic
metabolites, but also with damage to phospholipid and
free fatty acid molecules resulting in a decrease of their
levels.
The intensity of lipid peroxidation processes may be
detected by the determination of their intermediates and
end products. The most common are the determinations
of the level of thiobarbituric acid (TBA) reactive
substances (such as MDA), conjugated dienes and
hydroperoxides. The method in which the reaction of
MDA with TBA is involved, is based on spectrophotometric determination of pink product. Although the
reaction is rather sensitive, it is not only speci®c to
MDA (Bigwood and Read 1989). Very often the
presence of lipid peroxidation inhibitors such as 3,5
diisobutyl-4-hydroxytoluol (BHT) is necessary when
determinations of real MDA level are carried out. The
determination of intermediates such as hydroperoxides
is based on the reaction with KJ (potassium iodide) then
cadmium acetate and ®nally spectrophotometric detection at 353 nm (Ward et al. 1985). The presence of
conjugated double bonds in unsaturated fatty acids is
connected with lipid peroxidation processes. Such conjugated dienes can also be detected spectrophotometrically at 234 nm. The determination of these, as well as
hydroperoxides requires the extraction of lipids to avoid
turbidity of the sample.
Presented here, the three parameters represent di€erent steps of lipid peroxidation. Determinations of all
three parameters are necessary to provide a complete
description of the intensity of this process.
Protein peroxidation
Proteins consisting of amino acids are susceptible to
peroxidation processes caused by ROS (Bartosz 1995).
Protein peroxidation processes do not have chain
M Kankofer
character but protein peroxides which are created have
a rather long half-life ± about 36 h and may move far
away from the place of formation (Bartosz 1995).
Although protein peroxidation is not as ecient as lipid
peroxidation, it leads to the modi®cation of amino acid
residues, aggregation or fragmentation of protein molecules and the loss of biological activity. Peroxidative
damage of proteins is mainly caused by hydroxyl
radicals, but superoxide anion radicals and hydrogen
peroxide might also be involved (Bartosz 1995). The
thiol groups of cysteine are especially exposed to
peroxidative damage as are tyrosine, methionine, histidine and tryptophan. The thiol groups of cysteine are
oxidized to disulphide bridges, tryptophan to formylokinurenine and the recombination of tyrosine radicals
leads to the formation of bityrosine bridges (Goldstein
et al. 1994). There is evidence that proteins damaged by
peroxidative processes may more easily undergo proteolysis (Stadtman 1992). Such proteolysis might be the
restorative processes of proteins or the result of possible
denaturation caused by ROS (Bartosz 1995).
The consequence of protein peroxidative damage is
the inactivation of enzymes (Scherer and Deamer 1986)
and the loss of biological activity of proteins. Such
changes may lead to disturbances in metabolic pathways
and clinical symptoms of illnesses.
Protein peroxidative damage can be detected by the
determination of the levels of end products of the
reaction between ROS and aromatic amino acids such
as bityrosine (Goldstein et al. 1994) and formylokinurenine by spectro¯uorimetric methods. Tryptophan residue levels, which are destroyed under the in¯uence of
ROS, and amino groups that may be involved in the
reaction with aldehyde products of lipid peroxidation,
can also be determined spectro¯uorimetrically (RiceEvans et al. 1991). Spectrophotometric methods are
used for the detection of the level of thiol groups, which
are oxidized by ROS, and carbonyl groups, which serve
as markers of the oxidative modi®cation of proteins
(Rice-Evans et al. 1991; Goldstein et al. 1994).
As in the case of lipid peroxidation, the parameters
presented represent di€erent aspects of protein damage.
It is necessary to determine these parameters for a full
description of this process.
Nucleic acids peroxidation
Nucleic acids, like other biologically important macromolecules are also susceptible to oxidative damage
caused by ROS, although they are more stable than
lipids and proteins. This damage includes chemical
modi®cations of purines, pyrimidines and pentoses as
well as the breakdown of bonds between bases and
between nucleotides (Bartosz 1995; Box et al. 1995).
Singlet oxygen (Bartosz 1995) and hydroxyl radicals
(Chevion 1988) might be responsible for this damage.
Thymidine is the most susceptible to oxidative damage.
Its reaction with the hydroxyl radical may lead to the
creation of free radical of thymidine. This, in turn,
reacts with oxygen to form hydroperoxides. One of the
known metabolites of thymidine hydroperoxides is
thymidine glycol. Purine oxidative damage is based on
oxidation at di€erent carbon atoms. The most common
Bovine Placental Release/retention Processes and Peroxidative Damage
is C8-hydroxylation caused mainly by singlet oxygen
(Dizdaroglu 1991) producing 8-hydroxy-2¢-deoxyguanosine (8OH-dG) as a result. There are reports based on
in vitro experiments that the presence of 8OH-dG in
DNA may lead to transversions in purine±pyrimidine
bases and other mutations (Cheng et al. 1992). Thymidine glycol as well as 8OH-dG can be detected in urine,
serum and tissues, all of which indicate oxidative
damage to DNA (Loft et al. 1993).
Mammalian cells possess the mechanisms for recognizing DNA damage, as well as repair mechanisms. Any
repair activity is based on the action of DNA glycosylases and endonucleases (Demple and Harrison 1994;
Loft et al. 1994).
The consequences of oxidative damage of nucleic
acids are alterations in their structure leading to
improper protein biosynthesis. This, in turn, is re¯ected
in improper activity of di€erent enzymes and disturbances in metabolic pathways. As a result, clinical
symptoms of illnesses may occur.
The level of 8OH-dG, as the most common marker of
DNA oxidative damage, can be detected by thin layer
chromatography using 32P-post-labelling (Devanaboyina and Gupta 1996) as well as HPLC with electrochemical detection or gas chromatography±mass
spectrometry. All these methods require DNA extraction and enzymatic DNA digestion prior to chromatography. During analysis of the results, it is necessary to
consider the factors of age, sex (Bohr and Anson 1995)
and metabolic rate (Loft et al. 1993; Demple and
Harrison 1994) that may in¯uence the level of DNA
adducts.
Peroxidative processes and placental retention
Retention of fetal membranes in cows, as one of the
postpartum syndromes, is important not only because of
reproductive disorders of the mother and the health of
the newborn calf, but also because of economic losses.
Biochemical mechanisms responsible for the proper
release as well as the retention of fetal membranes still
require clari®cation. There are however, reports describing higher plasma progesterone and lower oestrogen
levels in cows a€ected by retention of fetal membranes
in comparison with control cows (Chew et al. 1972;
Grunert et al. 1989). Disturbances in triglycerides
(Kankofer et al. 1996a) and unsaturated fatty acids
(Kankofer et al. 1996b) as well as prostaglandins (Leidl
et al. 1980; Slama et al. 1993) also occur. The electrophoretic pattern of placental proteins is di€erent in
cases of retained and released fetal membranes (Maj and
Kankofer 1998).
Bearing in mind the indirect negative e€ects of ROS
on metabolic pathways, all the alterations in the levels of
the above-mentioned substances may be considered in
terms of being either the cause or result of the imbalance
between production and neutralization of ROS.
Some con®rmations are described by Miller et al.
(1993) who compared total plasma antioxidant activity
before parturition in cows retaining and releasing
placenta. This activity increased in the plasma of cows
that released the fetal membranes and decreased in those
with retained placenta. The activity of red blood cells
29
GSH-Px as well as the level of glutathione shortly before
parturition di€ered between animals retaining the placenta and control cows (BrzezinÂska-SÂlebodzinÂska et al.
1994). Shortly after parturition the activity of placental
GSH-Px, SOD (Kankofer et al. 1996c), GSH-Tr and
CAT (Kankofer 2001c) showed alterations between
retained and not-retained placenta. There are reports
describing compensatory and synergistic action between
antioxidative enzymes in the cells (Guemouri et al. 1991;
Michiels et al. 1994).
Indirect estimation of ROS, measured by the determination of parameters indicating the intensity of lipid
peroxidation, showed elevated levels of TBA reactive
substances, hydroperoxides and conjugated dienes in
retained placental tissues in comparison with control
cows (Kankofer 2001a). The parameters indicating the
intensity of protein peroxidation processes such as
formylokinurenine and bityrosine levels were higher in
animals with retained placenta than in those in which
the placenta was not retained. The concentrations of
the thiol groups showed the opposite relationship. The
levels of tryptophan were lower in retained placenta
than in control animals (Kankofer 2001b). The level of
8OH-dG, the parameter indicating the intensity of
DNA oxidative damage, was higher in retained than
not retained placenta of cows undergoing caesarian
section, but lower in retained placenta of spontaneously delivering animals (Kankofer and Schmerold,
submitted).
In conclusion, indirect ROS determination by estimation of the intensity of peroxidative processes of
biologically important macromolecules may be helpful
in the description of oxidative status during di€erent
physiological and pathological conditions. The imbalance between production and neutralization of ROS
seems to appear during the retention of fetal membranes
in cows. Whether this imbalance is the result or the
cause of retention still requires clari®cation and further
experiments.
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Submitted: 15. 01. 2001
Author's address: Marta Kankofer DVM, PhD, Department of
Biochemistry, Faculty of Veterinary Medicine, Agricultural University, 20±123 Lublin ul. Lubartowska 58 a Poland. E-mail: Kankofer@
agros.ar.lublin.pl