- Email: [email protected]
Antioxidant compounds and oxidative stress in female dogs during pregnancy
Research in Veterinary Science 83 (2007) 188–193 www.elsevier.com/locate/rvsc
Antioxidant compounds and oxidative stress in female dogs during pregnancy C.I. Vannucchi a
b
a,*
, A.A. Jordao b, H. Vannucchi
b
Department of Animal Reproduction, Faculty of Veterinary Medicine, University of Sa˜o Paulo, Av Prof Orlando Marques de Paiva, 87, Cidade Universita´ria, Sa˜o Paulo, SP, Brazil Nutrition and Metabolism, Department of Internal Medicine, Faculty of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, SP, Brazil Accepted 12 December 2006
Abstract Pregnancy is a physiological period during which different metabolic pathways are altered, resulting in greater oxygen consumption and modifications of the consumption of energy substrates, with a consequent greater exposure to oxidative stress. The objective of the present study was to determine and describe the serum profile of some antioxidant biomarkers and of oxidative stress markers during pregnancy in healthy dogs. Twenty nonpregnant (NP) and 20 pregnant (P) female dogs were studied. Serum samples were obtained from the animals during the 1st, 3rd, 5th and 7th weeks of pregnancy or during diestrus for the dosage of antioxidant molecules (vitamin E, vitamin A, zinc and magnesium) and oxidative stress markers (TBARS and carbonyl protein). The results revealed a statistically significant difference (p < 0.05) between the P and NP groups during the 3rd and 5th week for vitamins A and E (NP > P), and between the 1st and 3rd week for magnesium (NP > P). The other parameters did not differ between weeks within the same group or between groups. The present study shows that the levels of antioxidant molecules of pregnant dogs differed from that of nonpregnant dogs. These mechanisms may represent a protection against oxidative stress during this period for this species, a fact that definitely deserves further study. Also, the participation of other protective mechanisms and the interference of the fetal–placental unit with oxidative stress are still unknown. 2007 Elsevier Ltd. All rights reserved. Keywords: Antioxidants; Oxidative stress; Vitamins; Minerals; Pregnancy; Diestrus; Canine
1. Introduction The dynamic changes that occur in various body systems during the gestational period result in increased oxygen demand and changes in the consumption of the energy substrate by the mother, especially in the fetal–placental unit (Casanueva and Viteri, 2003). Different biochemical pathways that occur during pregnancy eventually lead to oxidative stress, which is represented by an imbalance between the formation of free radicals and the capacity for defense of the antioxidant mechanisms (Krieger and Loch-Caruso, 2001). When there is an excess of pro-oxidants and an extreme formation of free radicals, there is *
Corresponding author. Tel.: +55 11 3091 1423; fax: +55 11 3091 7412. E-mail address: [email protected] (C.I. Vannucchi).
0034-5288/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2006.12.009
a strong risk of DNA, lipid, and protein damage, which may lead to apoptosis (Miller et al., 1993; Poston and Raijmakers, 2004). For example, dietary deficiency of antioxidant nutrients and/or their limited body reserve during pregnancy can be an important cause of embryo resorption and abortion (Lee et al., 2004; Liurba et al., 2004; Poston and Raijmakers, 2004). Therefore, the acute oxidative stress generated during pregnancy has to be neutralized by potent antioxidants mechanisms in order to avoid such hazards. The antioxidant elements can limit the formation of free radicals through inhibition of lipid peroxidation and protein oxidation or by neutralizing their toxic effects (Little and Gladen, 1999). According to Evans and Halliwell (2001) the antioxidants defenses consist of low molecular mass antioxidants, intracellular enzymes, sequestration of
C.I. Vannucchi et al. / Research in Veterinary Science 83 (2007) 188–193
transition metal ions and repair mechanisms. Particularly important low molecular mass antioxidants are glutathione, vitamin E, ubiquinone, b-carotene, ascorbic acid (vitamin C), urate, and vitamin A. In addition, essential minerals such as zinc and magnesium are also required for the action of antioxidant enzymes (Osada et al., 2002; Miller et al., 1993). Fetal growth and development during pregnancy depend on the progressive maternal supply of essential nutrients such as vitamins and minerals (Baker et al., 2002). Thus, the increased maternal nutritional demands may alter the profile of the vitamins and minerals mobilized by the possible exacerbated oxidative process. On this basis, the maternal organism must develop a series of protective antioxidant mechanisms in order to allow for adequate progression of gestation. For example, although the fetal–placental unit is a site of high physiologic demand and therefore a source of reactive oxygen species, it possesses highly efficient protective mechanisms, especially liposoluble antioxidants such as vitamin E. In addition, through increasing placental transport, vitamins and minerals are removed from the maternal circulation in order to neutralize the formation of free radicals (Baker et al., 2002). In humans, Osada et al. (2002) demonstrated the occurrence of a constant afflux of copper and zinc from the maternal circulation to the fetus and a placental storage of magnesium and selenium in order to guarantee a constant supply to the fetal circulation. Although there is extensive data for humans, no reports about the mechanisms of protection during pregnancy are available to date for the canine species. Some studies have reported increased oxidative stress and reduced plasma concentrations of some antioxidants (such as vitamin E) in specific situations, such as repeated physical activity, during anesthetic induction and in dogs with immune-mediated hemolytic anemia or mammary tumours (Naziroglu and Gu¨nay, 1999; Hinchcliff et al., 2000; Pesillo et al., 2004; Kumaraguruparan et al., 2005). Despite the constant advances in the nutritional management of the species over the last 20 years, the nutritional requirements that will prevent damage and toxic effects during pregnancy in dogs are still unknown. The objective of the present study is to determine a broad-spectrum profile of some antioxidant elements (vitamin A, vitamin E, zinc and magnesium) and of the current utilized oxidative stress biomarkers (TBARS and carbonyl protein) during pregnancy in the canine species, and contribute to the future identification of vitamin and mineral deficiencies that might indicate metabolic stress during pregnancy. 2. Materials and methods 2.1. Animals The study was conducted on 40 clinically healthy Great Dane females in the reproductive age (range: 2–7 years),
189
with no history of infectious or inflammatory processes. The possibility of working with an exclusive breed minimizes the extreme variations that might exist among breeds. Animals presenting any clinical signs of renal, hepatic, intestinal or metabolic disease or those receiving any type of medication were excluded from the study due to the potential interference in oxidative stress. The animals’ health condition was assessed by a complete physical exam. A total white blood cell count was performed to help rule out any underlying inflammatory or infectious disease processes. All dogs received the same diet, consisting of commercial dog food for adult animals [Pedigree Advance Formula: crude protein (min.) 20%, crude fat (min.) 12%, crude fiber (max.) 5%, moisture (max.) 10%, ash (max.) 10.5%] and water was given ad libitum. Sanitary management was uniform, with all animals being helminth-free and periodically immunized. Serology for dirofilariosis and brucellosis was also performed routinely. The animals were divided into two groups: nonpregnant (NP) control group and pregnant (P) group, respectively, consisting of 20 females in diestrus and 20 pregnant females. Bitches were distributed among groups according to the same mean age. The beginning of diestrus was determined by vaginal cytology according to the method standardized by Olson et al. (1984). 2.2. Sample collection Blood was collected at every two week interval, starting at the first week after the beginning of the cytologic diestrus and continuing up to the 7th week of pregnancy, resulting in a total of four collections per animal (1st, 3rd, 5th and 7th weeks). On this basis, the antioxidant profile was obtained at two time points during the first half of pregnancy and at two time points during the final period of pregnancy. The animals fasted for 12 h prior to the collection of the blood samples. These were collected by puncturing the right or left saphenous vein using a vacuum blood collection system (Vacutainer Becton & Dickinson). After collection, the blood samples were immediately centrifuged at 1500g for 10 min. The serum obtained was divided into aliquots which were stored in plastic flasks resistant to freezing, carried to the laboratory and stored in a freezer at 70 C until processing. 2.3. Biochemical determinations The determination of thiobarbituric acid-reactive substances (TBARS) was used to estimate, using the method of Buege and Aust (1978), the degree of lipid peroxidation that had occurred during the period of evaluation. The determination of carbonyl protein as a general marker of the occurrence of protein oxidation was performed according to the method of Levine et al. (1990). Plasma vitamin A and E levels were determined by HPLC using a 4.6 · 25 cm C-18 type column (Shimpack
190
C.I. Vannucchi et al. / Research in Veterinary Science 83 (2007) 188–193
CLC-ODS) and a 4 mm · 1 cm pre-column, at a flow rate of 2.0 ml/min (Arnaud et al., 1991). Serum zinc and magnesium were measured using an atomic absorption spectrometer. In atomic absorption, the digested liquid sample is aspirated into a flame to produce free atoms of the element. The amount of light absorbed at the specific wavelength of the metal is directly proportional to the concentration of the element. 2.4. Statistical analysis
Throughout the experiment, the amount of vitamins A and E were always inferior in the pregnant group compared to the control group, as shown in Table 3 and Fig. 1. However, there was a significant difference (p < 0.05) between groups only during the 3rd and 5th weeks (about halfway through pregnancy). Table 3 Mean (±SD) values of serum vitamin A (lm) concentrations during the weeks of diestrus and pregnancy in female dogs Week
The data for the parameters evaluated in this study were first tested for normal distribution by the Kolmogorov– Smirnov test. For the variables shown to be parametric, the t-test for independent samples was used for vertical comparison (group NP · group P) and the t-test for paired samples was used for horizontal comparison (between weeks within the same group). Comparison of the mean values and analysis of variance were performed with the level of significance set at 5% (p < 0.05).
Vitamin A (lm) NP (diestrus)
P (pregnant)
a
1 3 5 7
1.52 ± 0.50a 1.45 ± 0.36a* 1.42 ± 0.43a* 1.55 ± 0.47a
1.87 ± 0.78 1.96 ± 0.66a 1.91 ± 0.72a 1.93 ± 0.73a
Superscript symbol * between columns on the same line indicates significant differences (p < 0.05). Different superscript letters within the same group in the same column indicate significant differences (p < 0.05).
25
3. Results
*
*
20
There was no difference between groups or between weeks within the same group regarding TBARS or carbonyl protein levels, as shown in Table 1. There was no statistically significant difference in serum zinc between groups or between weeks within the same group (Table 2). Table 2 also shows that serum magnesium levels were lower in the pregnant animals, compared to the group in diestrus, during the 1st and 3rd weeks (first half of pregnancy) (p < 0.05). Over the subsequent weeks, the values were similar for both groups.
Vit. E (μM/L)
15 Diestrus Pregnancy
10 5 0 1˚
*
3˚
5˚
7˚ Week
p<0.05
Fig. 1. Mean serum levels of vitamin E (lM/l) during the weeks of diestrus and during pregnancy in female dogs.
Table 1 Mean (±SD) serum TBARS and carbonyl protein concentrations during the weeks of diestrus and pregnancy in dogs Week
TBARS (nM/mg of protein) NP (diestrus)
1 3 5 7
Carbonyl protein (nM/mg of protein) P (pregnant)
a
NP (diestrus)
a
1.98 ± 0.76 2.32 ± 1.18a 2.45 ± 1.23a 2.23 ± 1.13a
P (pregnant)
a
2.39 ± 0.77 2.75 ± 1.22a 2.90 ± 1.16a 2.80 ± 0.99a
1.54 ± 0.86a 1.19 ± 0.67a 1.49 ± 0.69a 1.75 ± 1.04a
1.13 ± 0.73 0.97 ± 0.82a 1.40 ± 0.56a 1.37 ± 0.79a
Different superscript letters within the same group in the same column indicate significant differences (p < 0.05).
Table 2 Mean (±SD) values of serum zinc and magnesium during the weeks of diestrus and pregnancy in female dogs Week
Zinc (mg%)
Magnesium (mEq/l)
NP (diestrus) 1 3 5 7
P (pregnant) a
102.70 ± 26.17 98.34 ± 16.94a 101.87 ± 18.07a 104.12 ± 23.45a
NP (diestrus) a
90.21 ± 17.82 94.34 ± 24.70a 92.68 ± 19.24a 91.15 ± 16.57a
a
1.88 ± 0.22 1.95 ± 0.22a 1.83 ± 0.28a 1.85 ± 0.32a
P (pregnant) 1.63 ± 0.43a* 1.74 ± 0.14a* 1.87 ± 0.37a 1.73 ± 0.17a
Superscript symbol * between columns on the same line indicates significant differences (p < 0.05). Different superscript letters within the same group in the same column indicate significant differences (p < 0.05).
C.I. Vannucchi et al. / Research in Veterinary Science 83 (2007) 188–193
4. Discussion and conclusions Increased oxygen consumption in the cellular respiratory processes is expected to occur during gestation in order to achieve greater energy availability. For this reason, pregnancy is known to be a period in which increased oxidative stress occurs (Harma et al., 2004). However, in the present experiment there was no significant difference between pregnant females and females in diestrus regarding lipid peroxidation (TBARS) or protein oxidation (carbonyl protein) based on the markers evaluated. For this specific profile we speculate that the increased consumption of some antioxidant elements, such as vitamins A, E and magnesium might have neutralized the oxidative stress generated during pregnancy. Thus, we suggest that the expected increase in the oxidative stress biomarkers during pregnancy could have been controlled by efficient antioxidants mechanisms. Favoring this statement is the fact that the levels of vitamins E, A and magnesium were lower in the P group compared to the NP group, while the TBARS and carbonyl protein levels remained unchanged. According to Harma et al. (2004), oxidative stress and lipoperoxidation are higher in pregnant women, but so is antioxidant protection, when compared to nonpregnant women. Nevertheless, it is also possible that the decreased profiles of vitamins A, E and magnesium during pregnancy may be attributed to a greater consumption of these elements by the fetuses, without any relationship with the oxidative stress depletion. In this case, the antioxidant mechanisms mobilized during pregnancy would be related to other elements than the ones evaluated here. The detection of oxidative stress and free radicals can be performed by several methods according to the origin of the damage: cellular DNA, proteins, or lipids. Protein–carbonyls result from the interaction of free radicals with amino acid residues of oxidation. It is considered as an important marker of free radical damage (Casanueva and Viteri, 2003). In relation to lipid peroxidation, the most commonly used assay is the one that detects TBARS, although many of the aldehydes produced through lipid peroxidation can be used for this estimation (Casanueva and Viteri, 2003). In the present study, we opted to perform a broad analysis of free radical damage, through carbonyl protein and TBARS, however the possibility that other specific protein oxidation or lipid peroxidation indicators could have detected protein or lipid damage during pregnancy, respectively, cannot be ruled out. It is important to point out that lipid and protein oxidation may be restricted to specific tissues such as the fetal or placental tissue, a fact that, in general terms, may alter the blood concentrations of the major oxidative markers. Also, according to Liurba et al. (2004), the products of peroxidation are rapidly metabolized and detoxified. Therefore, elevated plasma concentrations of such products can usually be detected immediately after the oxidative challenge. Thus, the results of the present investigation do not exclude the occurrence of oxidation processes at restricted sites
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such as the placenta. In this respect, it is advisable to determine the indicators of oxidative stress directly at the target sites, such as the placental tissue or the umbilical cord blood. Mineral elements participate in the mechanism of defense against oxidative stress by being the constituents of various enzymes and regulators of enzymatic systems. Also, they are essential for organogenesis and tissue formation and therefore, their function in pregnancy is fundamental. In the present study, magnesium levels were statistically lower during the first half of pregnancy compared to nonpregnant females. This result may suggest a higher consumption of this mineral in the early stages of gestation, a period during which placentation ends, and organogenesis and tissue formation begins, generating a higher demand for this mineral by the placenta and fetuses. In addition, magnesium is one of the physiologic efficient antioxidants that hinder the oxidative processes (Evans and Halliwell, 2001). Therefore, its reduced plasma concentration in pregnant bitches can be explained by the activation of oxidative pathways by which it relates to magnesium. Regarding the serum zinc profile, no difference was observed between groups or between weeks within the same group. Although zinc plays an important role in different enzymatic systems in antioxidant defense, it appears to be mobilized during pregnancy in a less extent manner than the others antioxidants verified here for the canine species. Among all vitamin classes, vitamin A, vitamin C, and vitamin E are particularly important as protective elements against oxidative stress. Vitamin A is fundamental for reproductive and proliferative processes, especially for embryo and fetal development, in addition to having an antioxidant function, as it removes the free radicals formed during cellular respiration (Baker et al., 2002; Herrera et al., 2004). Placental transport of vitamin A has also been reported (Herrera et al., 2004). Therefore, preservation of its fetal concentration, despite decrease in the maternal circulation is of fundamental importance for the adequate course of gestation. In the present study, vitamin A concentrations in pregnant bitches were lower than for nonpregnant females up to the halfway point of pregnancy. This vitamin A profile supports the idea that there is a greater demand for this element due to its reallocation to placental and fetal metabolism, especially during the initial stages of pregnancy. An increase of production of free radicals in the oxidative stress during pregnancy stimulates the synthesis of prostaglandin F2a (PGF2a), a fact that might cause uterine contractions (Poston and Raijmakers, 2004). However, during pregnancy there is an inactivation of uterine PGF2a by trophoblastic proteins mainly by means of biotransformation into its inactive form, prostaglandin E2(PGE2). According to Napoli (1993), PGE2 is an antagonist of the retinoid function and therefore acts as a negative modulator of retinoic acid synthesis. On this basis, because of the greater production of PGE2 during pregnancy, there is an indirect reduction in vitamin A concentration as means
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C.I. Vannucchi et al. / Research in Veterinary Science 83 (2007) 188–193
of protection against eventual uterine contractions that might lead to abortion or to premature delivery. It is important to emphasize that the canine species presents a particular behavior regarding the metabolism of vitamin A: the preferential form of plasma transport is through retinyl esters and not through retinol, and dogs present a marked urinary excretion of retinol and retinyl palmitate which protects them against the possible toxic effects of vitamin A (Raila et al., 2002). For this reason, a more marked excretory loss is expected during pregnancy, since this period involves an increase in renal filtration rate (Benson and Thurmon, 1984). Thus the loss of vitamin A is even more marked, possibly leading to decrease in its serum concentration. Vitamin E is a potent protector against the formation of free radicals. It is considered to be a lipophilic antioxidant by inhibiting the lipid peroxidation of cell membranes, playing an important role in membrane stabilization (Qanungo et al., 1999). In women, the products of lipoperoxidation reach their highest levels in the second quarter of gestation, coinciding with the progressive elevation of plasma vitamin E concentrations (Lee et al., 2004). In the present experiment, an opposite profile was observed, i.e., approximately halfway through gestation vitamin E levels decreased (p < 0.05). This profile suggests that the oxidative stress generated during this period of gestation leads to a greater consumption of vitamin E. We believe the higher consumption of this element during the 3rd and 5th weeks of pregnancy may have influenced the stability of the marker of lipid peroxidation (TBARS) since the highest intensity of oxidative stress may be occurring during this period. In addition, as a lipophilic molecule which is soluble through the plasma membrane, vitamin E is highly incorporated into the placenta. Vitamin E is also concentrated in tissues that produce steroid hormones, such as the placenta, in order to protect the steroidogenic activities of the enzyme cytochrome P-450, which is highly sensitive to lipid peroxidation (Miller et al., 1993). In studies using vitamin E supplementation in cattle, Miller et al. (1993) demonstrated a more marked decrease in progesterone levels in non-supplemented cows in comparison to cows that received vitamin E during the peripartum period. On this basis, the reduction of plasma vitamin E observed during pregnancy in dogs may indicate an important placental sequestration of this element, since the blood concentrations of vitamin E were lower in pregnant females. Thus, the determination of vitamin E in placental tissues may elucidate this important question regarding the metabolism of this antioxidant during pregnancy in the canine species. The mechanism of action of vitamin E is the major factor responsible for the elimination of peroxyl radicals from lipid compartments, such as membranes or LDL, protecting them against oxidative damage. Thus, vitamin E concentration is directly correlated with lipid circulation, a fact that may confuse the interpretation of liposoluble antioxidant (Liurba et al., 2004). According to Herrera et al. (2004), vitamin E is responsible for an increase in oxidative
resistance of LDL and thus, may prevent the increase in oxidative stress on the basis of physiological hyperlipidemia towards the end of pregnancy. We showed here that the antioxidant profile of pregnant dogs differed from that of nonpregnant animals and no specific alterations were noted for the oxidative stress biomarkers selected. This statement could have addressed the conclusion of an efficient mechanism of protection against oxidative stress during pregnancy. However, the defense of the maternal organism against the damage caused by free radicals is provided by a broad-spectrum of elements acting synergistically. The present study describes the profile of some antioxidant mechanisms, a fact that definitely deserves further study. Thus, the participation of other protective mechanisms and the interference of oxidative stress within the fetal–placental unit are still unknown. It is known that control mechanisms of oxidative damage during canine pregnancy provide a supply of antioxidants to the developing fetuses, in preparation for extrauterine life. Unfortunately, to date no specific recommendations for pregnant dogs have been standardized concerning the nutrients evaluated in the present study; thus, the commercial diet available is assumed to fulfill daily requirements. Results clearly demonstrated a decrease of vitamin A, vitamin E and magnesium levels in the group of pregnant females. Future studies should determine daily requirements of these nutrients for domestic animals such as dogs, providing specific data for situations such as the gestational period. References Arnaud, J., Fortis, I., Blachier, S., Kia, D., Favier, A., 1991. Simultaneous determination of retinol, a-tocopherol and b-carotene in serum by isocratic high performance liquid chromatography. Journal of Chromatography 572, 103–116. Baker, H., DeAngelis, B., Holland, B., Gittens-Williams, L., Barrett Jr., T., 2002. Vitamin profile of 563 gravidas during trimesters of pregnancy. Journal of the American College of Nutrition 21, 33–37. Benson, G.J., Thurmon, J.C., 1984. Anesthesia for cesarian section in the dog and cat. Modern Veterinary Practice 30, 29–32. Buege, J.A., Aust, S.D., 1978. Microsomal lipid peroxidation. Methods in Enzymology 52, 302–310. Casanueva, E., Viteri, F.E., 2003. Iron and oxidative stress in pregnancy. Journal of Nutrition 133, 1700S–1708S. Evans, P., Halliwell, B., 2001. Micronutrients: oxidant/antioxidant status. British Journal of Nutrition 85, S67–S74. Harma, M., Harma, M., Kocyigit, A., 2004. Comparison of protein carbonyl and total plasma thiol concentrations in patients with complete hydatidiform mole with those in healthy pregnant women. Acta Obstetricia et Gynecologica Scandinavica 83, 857–860. Herrera, E., Ortega, H., Alvino, G., Giovannini, N., Amusquivar, E., Cetin, I., 2004. Relationship between plasma fatty acid profile and antioxidant vitamins during normal pregnancy. European Journal of Clinical Nutrition 58, 1231–1238. Hinchcliff, K.W., Reinhart, G.A., DiSilvestro, R., Reynolds, A., BlosteinFujii, A., Swenson, R.A., 2000. Oxidant stress in sled dogs subjected to repetitive endurance exercise. American Journal of Veterinary Research 61, 512–517. Krieger, T.R., Loch-Caruso, R., 2001. Antioxidants prevent c-hexachlorocyclohexane-induced inhibition of rat myometrial gap junctions and contractions. Biology of Reproduction 64, 537–547.
C.I. Vannucchi et al. / Research in Veterinary Science 83 (2007) 188–193 Kumaraguruparan, R., Balachandran, C., Murali Manohar, B., Nagini, S., 2005. Altered oxidant–antioxidant profile in canine mammary tumours. Veterinary Research Communications 29, 287–296. Lee, B.E., Hong, Y.C., Lee, K.H., Kim, Y.J., Kim, W.K., Chang, N.S., Park, E.A., Park, H.S., Hann, H.J., 2004. Influence of maternal serum levels of vitamins C and E during the second trimester on birth weight and length. European Journal of Clinical Nutrition 58, 1365–1371. Levine, R.L., Garland, D., Oliver, C.N., Amici, A., Climent, I., Lenz, A.G., Ahn, B.W., Shaltiel, S., Stadtman, E.R., 1990. Determination of carbonyl content in oxidatively modified proteins. Methods in Enzymology 186, 464–478. Little, R.E., Gladen, B.C., 1999. Levels of lipid peroxides in uncomplicated pregnancy: a review of the literature. Reproductive Toxicology 13, 347–352. Liurba, E., Grataco´s, E., Martin-Galla´n, P., Cabero, L., Domı´nguez, C., 2004. A comprehensive study of oxidative stress and antioxidant status in preeclampsia and normal pregnancy. Free Radical Biology and Medicine 37, 557–570. Miller, J.K., Brzezinska-Slebodzinska, E., Madsen, F.C., 1993. Oxidative stress, antioxidants, and animal function. Journal of Dairy Science 76, 2812–2823. Napoli, J.L., 1993. Prostaglandin E and 12-O-tetradecanoylphorbol-13acetate are negative modulators of retinoic acid synthesis. Archives of Biochemistry and Biophysics 300, 577–581.
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Naziroglu, M., Gu¨nay, C., 1999. The levels of some antioxidant vitamins, glutathione peroxidase and lipoperoxidase during the anaesthesia of dogs. Cell Biochemistry and Function 17, 207–212. Olson, P.N., Thrall, M.A., Nykes, P.M., Nett, T.M., 1984. Vaginal cytology. Part I. A useful tool for staging the canine estrous cycle. Compendium on Continuing Education for the Practicing Veterinarian 6, 288–297. Osada, H., Watanabe, Y., Nishimura, Y., Yukawa, M., Seki, K., Sekiya, S., 2002. Profile of trace element concentrations in the feto-placental unit in relation to fetal growth. Acta Obstetrica et Gynecologica Scandinavica 81, 931–937. Pesillo, S.A., Freeman, L.M., Rush, J.E., 2004. Assessment of lipid peroxidation and serum vitamin E concentration in dogs with immunemediated hemolytic anemia. American Journal of Veterinary Research 65, 1621–1624. Poston, L., Raijmakers, T.M., 2004. Trophoblast oxidative stress, antioxidants and pregnancy outcome – a review. Placenta 25 (Suppl. A), S72–S78. Qanungo, S., Sen, A., Mukherjea, M., 1999. Antioxidant status and lipid peroxidation in human feto-placental unit. Clnica Chimica Acta 285, 1–12. Raila, J., Radon, R., Trupschuch, A., Schweigert, F.J., 2002. Retinol and retinyl ester responses in the blood plasma and urine of dogs after a single oral dose of vitamin A. Journal of Nutrition 132 (Suppl. 2), 1673S–1675S.
Antioxidant compounds and oxidative stress in female dogs during pregnancy C.I. Vannucchi a
b
a,*
, A.A. Jordao b, H. Vannucchi
b
Department of Animal Reproduction, Faculty of Veterinary Medicine, University of Sa˜o Paulo, Av Prof Orlando Marques de Paiva, 87, Cidade Universita´ria, Sa˜o Paulo, SP, Brazil Nutrition and Metabolism, Department of Internal Medicine, Faculty of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, SP, Brazil Accepted 12 December 2006
Abstract Pregnancy is a physiological period during which different metabolic pathways are altered, resulting in greater oxygen consumption and modifications of the consumption of energy substrates, with a consequent greater exposure to oxidative stress. The objective of the present study was to determine and describe the serum profile of some antioxidant biomarkers and of oxidative stress markers during pregnancy in healthy dogs. Twenty nonpregnant (NP) and 20 pregnant (P) female dogs were studied. Serum samples were obtained from the animals during the 1st, 3rd, 5th and 7th weeks of pregnancy or during diestrus for the dosage of antioxidant molecules (vitamin E, vitamin A, zinc and magnesium) and oxidative stress markers (TBARS and carbonyl protein). The results revealed a statistically significant difference (p < 0.05) between the P and NP groups during the 3rd and 5th week for vitamins A and E (NP > P), and between the 1st and 3rd week for magnesium (NP > P). The other parameters did not differ between weeks within the same group or between groups. The present study shows that the levels of antioxidant molecules of pregnant dogs differed from that of nonpregnant dogs. These mechanisms may represent a protection against oxidative stress during this period for this species, a fact that definitely deserves further study. Also, the participation of other protective mechanisms and the interference of the fetal–placental unit with oxidative stress are still unknown. 2007 Elsevier Ltd. All rights reserved. Keywords: Antioxidants; Oxidative stress; Vitamins; Minerals; Pregnancy; Diestrus; Canine
1. Introduction The dynamic changes that occur in various body systems during the gestational period result in increased oxygen demand and changes in the consumption of the energy substrate by the mother, especially in the fetal–placental unit (Casanueva and Viteri, 2003). Different biochemical pathways that occur during pregnancy eventually lead to oxidative stress, which is represented by an imbalance between the formation of free radicals and the capacity for defense of the antioxidant mechanisms (Krieger and Loch-Caruso, 2001). When there is an excess of pro-oxidants and an extreme formation of free radicals, there is *
Corresponding author. Tel.: +55 11 3091 1423; fax: +55 11 3091 7412. E-mail address: [email protected] (C.I. Vannucchi).
0034-5288/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2006.12.009
a strong risk of DNA, lipid, and protein damage, which may lead to apoptosis (Miller et al., 1993; Poston and Raijmakers, 2004). For example, dietary deficiency of antioxidant nutrients and/or their limited body reserve during pregnancy can be an important cause of embryo resorption and abortion (Lee et al., 2004; Liurba et al., 2004; Poston and Raijmakers, 2004). Therefore, the acute oxidative stress generated during pregnancy has to be neutralized by potent antioxidants mechanisms in order to avoid such hazards. The antioxidant elements can limit the formation of free radicals through inhibition of lipid peroxidation and protein oxidation or by neutralizing their toxic effects (Little and Gladen, 1999). According to Evans and Halliwell (2001) the antioxidants defenses consist of low molecular mass antioxidants, intracellular enzymes, sequestration of
C.I. Vannucchi et al. / Research in Veterinary Science 83 (2007) 188–193
transition metal ions and repair mechanisms. Particularly important low molecular mass antioxidants are glutathione, vitamin E, ubiquinone, b-carotene, ascorbic acid (vitamin C), urate, and vitamin A. In addition, essential minerals such as zinc and magnesium are also required for the action of antioxidant enzymes (Osada et al., 2002; Miller et al., 1993). Fetal growth and development during pregnancy depend on the progressive maternal supply of essential nutrients such as vitamins and minerals (Baker et al., 2002). Thus, the increased maternal nutritional demands may alter the profile of the vitamins and minerals mobilized by the possible exacerbated oxidative process. On this basis, the maternal organism must develop a series of protective antioxidant mechanisms in order to allow for adequate progression of gestation. For example, although the fetal–placental unit is a site of high physiologic demand and therefore a source of reactive oxygen species, it possesses highly efficient protective mechanisms, especially liposoluble antioxidants such as vitamin E. In addition, through increasing placental transport, vitamins and minerals are removed from the maternal circulation in order to neutralize the formation of free radicals (Baker et al., 2002). In humans, Osada et al. (2002) demonstrated the occurrence of a constant afflux of copper and zinc from the maternal circulation to the fetus and a placental storage of magnesium and selenium in order to guarantee a constant supply to the fetal circulation. Although there is extensive data for humans, no reports about the mechanisms of protection during pregnancy are available to date for the canine species. Some studies have reported increased oxidative stress and reduced plasma concentrations of some antioxidants (such as vitamin E) in specific situations, such as repeated physical activity, during anesthetic induction and in dogs with immune-mediated hemolytic anemia or mammary tumours (Naziroglu and Gu¨nay, 1999; Hinchcliff et al., 2000; Pesillo et al., 2004; Kumaraguruparan et al., 2005). Despite the constant advances in the nutritional management of the species over the last 20 years, the nutritional requirements that will prevent damage and toxic effects during pregnancy in dogs are still unknown. The objective of the present study is to determine a broad-spectrum profile of some antioxidant elements (vitamin A, vitamin E, zinc and magnesium) and of the current utilized oxidative stress biomarkers (TBARS and carbonyl protein) during pregnancy in the canine species, and contribute to the future identification of vitamin and mineral deficiencies that might indicate metabolic stress during pregnancy. 2. Materials and methods 2.1. Animals The study was conducted on 40 clinically healthy Great Dane females in the reproductive age (range: 2–7 years),
189
with no history of infectious or inflammatory processes. The possibility of working with an exclusive breed minimizes the extreme variations that might exist among breeds. Animals presenting any clinical signs of renal, hepatic, intestinal or metabolic disease or those receiving any type of medication were excluded from the study due to the potential interference in oxidative stress. The animals’ health condition was assessed by a complete physical exam. A total white blood cell count was performed to help rule out any underlying inflammatory or infectious disease processes. All dogs received the same diet, consisting of commercial dog food for adult animals [Pedigree Advance Formula: crude protein (min.) 20%, crude fat (min.) 12%, crude fiber (max.) 5%, moisture (max.) 10%, ash (max.) 10.5%] and water was given ad libitum. Sanitary management was uniform, with all animals being helminth-free and periodically immunized. Serology for dirofilariosis and brucellosis was also performed routinely. The animals were divided into two groups: nonpregnant (NP) control group and pregnant (P) group, respectively, consisting of 20 females in diestrus and 20 pregnant females. Bitches were distributed among groups according to the same mean age. The beginning of diestrus was determined by vaginal cytology according to the method standardized by Olson et al. (1984). 2.2. Sample collection Blood was collected at every two week interval, starting at the first week after the beginning of the cytologic diestrus and continuing up to the 7th week of pregnancy, resulting in a total of four collections per animal (1st, 3rd, 5th and 7th weeks). On this basis, the antioxidant profile was obtained at two time points during the first half of pregnancy and at two time points during the final period of pregnancy. The animals fasted for 12 h prior to the collection of the blood samples. These were collected by puncturing the right or left saphenous vein using a vacuum blood collection system (Vacutainer Becton & Dickinson). After collection, the blood samples were immediately centrifuged at 1500g for 10 min. The serum obtained was divided into aliquots which were stored in plastic flasks resistant to freezing, carried to the laboratory and stored in a freezer at 70 C until processing. 2.3. Biochemical determinations The determination of thiobarbituric acid-reactive substances (TBARS) was used to estimate, using the method of Buege and Aust (1978), the degree of lipid peroxidation that had occurred during the period of evaluation. The determination of carbonyl protein as a general marker of the occurrence of protein oxidation was performed according to the method of Levine et al. (1990). Plasma vitamin A and E levels were determined by HPLC using a 4.6 · 25 cm C-18 type column (Shimpack
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CLC-ODS) and a 4 mm · 1 cm pre-column, at a flow rate of 2.0 ml/min (Arnaud et al., 1991). Serum zinc and magnesium were measured using an atomic absorption spectrometer. In atomic absorption, the digested liquid sample is aspirated into a flame to produce free atoms of the element. The amount of light absorbed at the specific wavelength of the metal is directly proportional to the concentration of the element. 2.4. Statistical analysis
Throughout the experiment, the amount of vitamins A and E were always inferior in the pregnant group compared to the control group, as shown in Table 3 and Fig. 1. However, there was a significant difference (p < 0.05) between groups only during the 3rd and 5th weeks (about halfway through pregnancy). Table 3 Mean (±SD) values of serum vitamin A (lm) concentrations during the weeks of diestrus and pregnancy in female dogs Week
The data for the parameters evaluated in this study were first tested for normal distribution by the Kolmogorov– Smirnov test. For the variables shown to be parametric, the t-test for independent samples was used for vertical comparison (group NP · group P) and the t-test for paired samples was used for horizontal comparison (between weeks within the same group). Comparison of the mean values and analysis of variance were performed with the level of significance set at 5% (p < 0.05).
Vitamin A (lm) NP (diestrus)
P (pregnant)
a
1 3 5 7
1.52 ± 0.50a 1.45 ± 0.36a* 1.42 ± 0.43a* 1.55 ± 0.47a
1.87 ± 0.78 1.96 ± 0.66a 1.91 ± 0.72a 1.93 ± 0.73a
Superscript symbol * between columns on the same line indicates significant differences (p < 0.05). Different superscript letters within the same group in the same column indicate significant differences (p < 0.05).
25
3. Results
*
*
20
There was no difference between groups or between weeks within the same group regarding TBARS or carbonyl protein levels, as shown in Table 1. There was no statistically significant difference in serum zinc between groups or between weeks within the same group (Table 2). Table 2 also shows that serum magnesium levels were lower in the pregnant animals, compared to the group in diestrus, during the 1st and 3rd weeks (first half of pregnancy) (p < 0.05). Over the subsequent weeks, the values were similar for both groups.
Vit. E (μM/L)
15 Diestrus Pregnancy
10 5 0 1˚
*
3˚
5˚
7˚ Week
p<0.05
Fig. 1. Mean serum levels of vitamin E (lM/l) during the weeks of diestrus and during pregnancy in female dogs.
Table 1 Mean (±SD) serum TBARS and carbonyl protein concentrations during the weeks of diestrus and pregnancy in dogs Week
TBARS (nM/mg of protein) NP (diestrus)
1 3 5 7
Carbonyl protein (nM/mg of protein) P (pregnant)
a
NP (diestrus)
a
1.98 ± 0.76 2.32 ± 1.18a 2.45 ± 1.23a 2.23 ± 1.13a
P (pregnant)
a
2.39 ± 0.77 2.75 ± 1.22a 2.90 ± 1.16a 2.80 ± 0.99a
1.54 ± 0.86a 1.19 ± 0.67a 1.49 ± 0.69a 1.75 ± 1.04a
1.13 ± 0.73 0.97 ± 0.82a 1.40 ± 0.56a 1.37 ± 0.79a
Different superscript letters within the same group in the same column indicate significant differences (p < 0.05).
Table 2 Mean (±SD) values of serum zinc and magnesium during the weeks of diestrus and pregnancy in female dogs Week
Zinc (mg%)
Magnesium (mEq/l)
NP (diestrus) 1 3 5 7
P (pregnant) a
102.70 ± 26.17 98.34 ± 16.94a 101.87 ± 18.07a 104.12 ± 23.45a
NP (diestrus) a
90.21 ± 17.82 94.34 ± 24.70a 92.68 ± 19.24a 91.15 ± 16.57a
a
1.88 ± 0.22 1.95 ± 0.22a 1.83 ± 0.28a 1.85 ± 0.32a
P (pregnant) 1.63 ± 0.43a* 1.74 ± 0.14a* 1.87 ± 0.37a 1.73 ± 0.17a
Superscript symbol * between columns on the same line indicates significant differences (p < 0.05). Different superscript letters within the same group in the same column indicate significant differences (p < 0.05).
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4. Discussion and conclusions Increased oxygen consumption in the cellular respiratory processes is expected to occur during gestation in order to achieve greater energy availability. For this reason, pregnancy is known to be a period in which increased oxidative stress occurs (Harma et al., 2004). However, in the present experiment there was no significant difference between pregnant females and females in diestrus regarding lipid peroxidation (TBARS) or protein oxidation (carbonyl protein) based on the markers evaluated. For this specific profile we speculate that the increased consumption of some antioxidant elements, such as vitamins A, E and magnesium might have neutralized the oxidative stress generated during pregnancy. Thus, we suggest that the expected increase in the oxidative stress biomarkers during pregnancy could have been controlled by efficient antioxidants mechanisms. Favoring this statement is the fact that the levels of vitamins E, A and magnesium were lower in the P group compared to the NP group, while the TBARS and carbonyl protein levels remained unchanged. According to Harma et al. (2004), oxidative stress and lipoperoxidation are higher in pregnant women, but so is antioxidant protection, when compared to nonpregnant women. Nevertheless, it is also possible that the decreased profiles of vitamins A, E and magnesium during pregnancy may be attributed to a greater consumption of these elements by the fetuses, without any relationship with the oxidative stress depletion. In this case, the antioxidant mechanisms mobilized during pregnancy would be related to other elements than the ones evaluated here. The detection of oxidative stress and free radicals can be performed by several methods according to the origin of the damage: cellular DNA, proteins, or lipids. Protein–carbonyls result from the interaction of free radicals with amino acid residues of oxidation. It is considered as an important marker of free radical damage (Casanueva and Viteri, 2003). In relation to lipid peroxidation, the most commonly used assay is the one that detects TBARS, although many of the aldehydes produced through lipid peroxidation can be used for this estimation (Casanueva and Viteri, 2003). In the present study, we opted to perform a broad analysis of free radical damage, through carbonyl protein and TBARS, however the possibility that other specific protein oxidation or lipid peroxidation indicators could have detected protein or lipid damage during pregnancy, respectively, cannot be ruled out. It is important to point out that lipid and protein oxidation may be restricted to specific tissues such as the fetal or placental tissue, a fact that, in general terms, may alter the blood concentrations of the major oxidative markers. Also, according to Liurba et al. (2004), the products of peroxidation are rapidly metabolized and detoxified. Therefore, elevated plasma concentrations of such products can usually be detected immediately after the oxidative challenge. Thus, the results of the present investigation do not exclude the occurrence of oxidation processes at restricted sites
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such as the placenta. In this respect, it is advisable to determine the indicators of oxidative stress directly at the target sites, such as the placental tissue or the umbilical cord blood. Mineral elements participate in the mechanism of defense against oxidative stress by being the constituents of various enzymes and regulators of enzymatic systems. Also, they are essential for organogenesis and tissue formation and therefore, their function in pregnancy is fundamental. In the present study, magnesium levels were statistically lower during the first half of pregnancy compared to nonpregnant females. This result may suggest a higher consumption of this mineral in the early stages of gestation, a period during which placentation ends, and organogenesis and tissue formation begins, generating a higher demand for this mineral by the placenta and fetuses. In addition, magnesium is one of the physiologic efficient antioxidants that hinder the oxidative processes (Evans and Halliwell, 2001). Therefore, its reduced plasma concentration in pregnant bitches can be explained by the activation of oxidative pathways by which it relates to magnesium. Regarding the serum zinc profile, no difference was observed between groups or between weeks within the same group. Although zinc plays an important role in different enzymatic systems in antioxidant defense, it appears to be mobilized during pregnancy in a less extent manner than the others antioxidants verified here for the canine species. Among all vitamin classes, vitamin A, vitamin C, and vitamin E are particularly important as protective elements against oxidative stress. Vitamin A is fundamental for reproductive and proliferative processes, especially for embryo and fetal development, in addition to having an antioxidant function, as it removes the free radicals formed during cellular respiration (Baker et al., 2002; Herrera et al., 2004). Placental transport of vitamin A has also been reported (Herrera et al., 2004). Therefore, preservation of its fetal concentration, despite decrease in the maternal circulation is of fundamental importance for the adequate course of gestation. In the present study, vitamin A concentrations in pregnant bitches were lower than for nonpregnant females up to the halfway point of pregnancy. This vitamin A profile supports the idea that there is a greater demand for this element due to its reallocation to placental and fetal metabolism, especially during the initial stages of pregnancy. An increase of production of free radicals in the oxidative stress during pregnancy stimulates the synthesis of prostaglandin F2a (PGF2a), a fact that might cause uterine contractions (Poston and Raijmakers, 2004). However, during pregnancy there is an inactivation of uterine PGF2a by trophoblastic proteins mainly by means of biotransformation into its inactive form, prostaglandin E2(PGE2). According to Napoli (1993), PGE2 is an antagonist of the retinoid function and therefore acts as a negative modulator of retinoic acid synthesis. On this basis, because of the greater production of PGE2 during pregnancy, there is an indirect reduction in vitamin A concentration as means
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of protection against eventual uterine contractions that might lead to abortion or to premature delivery. It is important to emphasize that the canine species presents a particular behavior regarding the metabolism of vitamin A: the preferential form of plasma transport is through retinyl esters and not through retinol, and dogs present a marked urinary excretion of retinol and retinyl palmitate which protects them against the possible toxic effects of vitamin A (Raila et al., 2002). For this reason, a more marked excretory loss is expected during pregnancy, since this period involves an increase in renal filtration rate (Benson and Thurmon, 1984). Thus the loss of vitamin A is even more marked, possibly leading to decrease in its serum concentration. Vitamin E is a potent protector against the formation of free radicals. It is considered to be a lipophilic antioxidant by inhibiting the lipid peroxidation of cell membranes, playing an important role in membrane stabilization (Qanungo et al., 1999). In women, the products of lipoperoxidation reach their highest levels in the second quarter of gestation, coinciding with the progressive elevation of plasma vitamin E concentrations (Lee et al., 2004). In the present experiment, an opposite profile was observed, i.e., approximately halfway through gestation vitamin E levels decreased (p < 0.05). This profile suggests that the oxidative stress generated during this period of gestation leads to a greater consumption of vitamin E. We believe the higher consumption of this element during the 3rd and 5th weeks of pregnancy may have influenced the stability of the marker of lipid peroxidation (TBARS) since the highest intensity of oxidative stress may be occurring during this period. In addition, as a lipophilic molecule which is soluble through the plasma membrane, vitamin E is highly incorporated into the placenta. Vitamin E is also concentrated in tissues that produce steroid hormones, such as the placenta, in order to protect the steroidogenic activities of the enzyme cytochrome P-450, which is highly sensitive to lipid peroxidation (Miller et al., 1993). In studies using vitamin E supplementation in cattle, Miller et al. (1993) demonstrated a more marked decrease in progesterone levels in non-supplemented cows in comparison to cows that received vitamin E during the peripartum period. On this basis, the reduction of plasma vitamin E observed during pregnancy in dogs may indicate an important placental sequestration of this element, since the blood concentrations of vitamin E were lower in pregnant females. Thus, the determination of vitamin E in placental tissues may elucidate this important question regarding the metabolism of this antioxidant during pregnancy in the canine species. The mechanism of action of vitamin E is the major factor responsible for the elimination of peroxyl radicals from lipid compartments, such as membranes or LDL, protecting them against oxidative damage. Thus, vitamin E concentration is directly correlated with lipid circulation, a fact that may confuse the interpretation of liposoluble antioxidant (Liurba et al., 2004). According to Herrera et al. (2004), vitamin E is responsible for an increase in oxidative
resistance of LDL and thus, may prevent the increase in oxidative stress on the basis of physiological hyperlipidemia towards the end of pregnancy. We showed here that the antioxidant profile of pregnant dogs differed from that of nonpregnant animals and no specific alterations were noted for the oxidative stress biomarkers selected. This statement could have addressed the conclusion of an efficient mechanism of protection against oxidative stress during pregnancy. However, the defense of the maternal organism against the damage caused by free radicals is provided by a broad-spectrum of elements acting synergistically. The present study describes the profile of some antioxidant mechanisms, a fact that definitely deserves further study. Thus, the participation of other protective mechanisms and the interference of oxidative stress within the fetal–placental unit are still unknown. It is known that control mechanisms of oxidative damage during canine pregnancy provide a supply of antioxidants to the developing fetuses, in preparation for extrauterine life. Unfortunately, to date no specific recommendations for pregnant dogs have been standardized concerning the nutrients evaluated in the present study; thus, the commercial diet available is assumed to fulfill daily requirements. Results clearly demonstrated a decrease of vitamin A, vitamin E and magnesium levels in the group of pregnant females. Future studies should determine daily requirements of these nutrients for domestic animals such as dogs, providing specific data for situations such as the gestational period. References Arnaud, J., Fortis, I., Blachier, S., Kia, D., Favier, A., 1991. Simultaneous determination of retinol, a-tocopherol and b-carotene in serum by isocratic high performance liquid chromatography. Journal of Chromatography 572, 103–116. Baker, H., DeAngelis, B., Holland, B., Gittens-Williams, L., Barrett Jr., T., 2002. Vitamin profile of 563 gravidas during trimesters of pregnancy. Journal of the American College of Nutrition 21, 33–37. Benson, G.J., Thurmon, J.C., 1984. Anesthesia for cesarian section in the dog and cat. Modern Veterinary Practice 30, 29–32. Buege, J.A., Aust, S.D., 1978. Microsomal lipid peroxidation. Methods in Enzymology 52, 302–310. Casanueva, E., Viteri, F.E., 2003. Iron and oxidative stress in pregnancy. Journal of Nutrition 133, 1700S–1708S. Evans, P., Halliwell, B., 2001. Micronutrients: oxidant/antioxidant status. British Journal of Nutrition 85, S67–S74. Harma, M., Harma, M., Kocyigit, A., 2004. Comparison of protein carbonyl and total plasma thiol concentrations in patients with complete hydatidiform mole with those in healthy pregnant women. Acta Obstetricia et Gynecologica Scandinavica 83, 857–860. Herrera, E., Ortega, H., Alvino, G., Giovannini, N., Amusquivar, E., Cetin, I., 2004. Relationship between plasma fatty acid profile and antioxidant vitamins during normal pregnancy. European Journal of Clinical Nutrition 58, 1231–1238. Hinchcliff, K.W., Reinhart, G.A., DiSilvestro, R., Reynolds, A., BlosteinFujii, A., Swenson, R.A., 2000. Oxidant stress in sled dogs subjected to repetitive endurance exercise. American Journal of Veterinary Research 61, 512–517. Krieger, T.R., Loch-Caruso, R., 2001. Antioxidants prevent c-hexachlorocyclohexane-induced inhibition of rat myometrial gap junctions and contractions. Biology of Reproduction 64, 537–547.
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