Experimental Gerontology 50 (2014) 45–51 Contents lists available at ScienceDirect Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero Age-related changes in ADMA–DDAH–NO pathway in rat liver subjected to partial ischemia followed by global reperfusion Małgorzata Trocha a, Anna Merwid-Ląd a, Ewa Chlebda-Sieragowska a, Andrzej Szuba b,c, Małgorzata Pieśniewska a, Lidia Fereniec-Gołębiewska a, Joanna Kwiatkowska a, Adam Szeląg a, and Tomasz Sozański a,⁎ a b c Department of Pharmacology, Wrocław Medical University, Mikulicza-Radeckiego 2, PL 50-345 Wrocław, Poland Department of Internal Medicine, 4th Military Hospital with Policlinic in Wrocław, Weigla 5, PL 50-981 Wrocław, Poland The Faculty of Health Science, Wrocław Medical University, Bartla 5, PL 50-996 Wrocław, Poland a r t i c l e i n f o Article history: Received 12 May 2013 Received in revised form 6 November 2013 Accepted 12 November 2013 Available online 20 November 2013 Section Editor: Andrzej Bartke Keywords: Age Liver Ischemia/reperfusion iNOS ADMA Rat a b s t r a c t Background: Liver function is affected during ischemia/reperfusion (IR). We evaluated the effect of the aging process on selected parameters determining the NO level in rat liver subjected to IR. Methods: The animals were divided into the C-2 and the IR-2 group of young rats (2–4 months old) and the C-12 and the IR-12 group of older rats (12–14 months old). Livers belonging to the IR-2 and the IR-12 group were subjected to partial ischemia (60 min) and reperfusion (4 h). Blood samples were obtained after surgeries to estimate the activity of aminotransferases, as well as just before ischemia and during reperfusion (15, 120, and 240 min) to estimate concentration of arginine (Arg) and its derivatives: asymmetric and symmetric dimethylarginine (ADMA, SDMA). After IR, dimethylarginine dimethylaminohydrolase (DDAH) activity and protein concentration of inducible nitric oxide synthase (iNOS) were measured in liver homogenates. Results: In the IR-2 group ADMA level increased the most between 15 and 120 min of reperfusion and was the highest of all the groups (0.72 ± 0.2 μmol/l). In the IR-12 group ADMA level decreased signiﬁcantly and was lower compared to all the other groups at 15 min (0.42 ± 0.2 μmol/l) and to IR-2 at 120 (0.52 ± 0.1 μmol/l) and 240 min (0.38 ± 0.1 μmol/l) of reperfusion. Only the IR-2 group SDMA level increased signiﬁcantly between 15 (0.75 ± 0.9 μmol/l) and 240 min (1.0 ± 1.2 μmol/l) of reperfusion. At the beginning of the surgery the Arg level was signiﬁcantly higher in young rats (C-2: 102.1 ± 35.7 μmol/l; IR-2: 114.63 ± 28.9 μmol/l) than in older ones (C-12: 41.88 ± 44.7 μmol/l; IR-12: 28.64 ± 30.6 μmol/l). In the C-2 group the Arg level (77.41 ± 37.5 μmol/l) and Arg/ADMA (A/A) ratio (138.03 ± 62.8 μmol/l) were signiﬁcantly higher compared to the ischemic groups at 15 min and to all the other groups at 120 (Arg: 47.17 ± 31.7 μmol/l; A/A: 88.28 ± 66.2 μmol/l) and 240 min (Arg: 43.87 ± 21.9 μmol/l; A/A: 118.02 ± 106.3 μmol/l). In the IR-2 group Arg level (11.4 ± 12.0 μmol/l) and A/A ratio (16.11 ± 16.2 μmol/l) decreased signiﬁcantly at 15 min and during the next phase of reperfusion the levels of those parameters were low, comparably to those in IR-12. As a result of IR, a decrease in DDAH activity and an increase in iNOS protein concentration were observed only in the young rats. Conclusions: We found that in the non-ischemic groups the Arg level may be affected by the aging process. Under IR conditions, important changes in DDAH–ADMA–NO pathway were observed only in young livers. © 2013 Elsevier Inc. All rights reserved. 1. Introduction Abbreviations: A/A ratio, arginine/ADMA ratio; ADMA, asymmetric dimethylarginine; ALT, alanine aminotransferase; Arg, arginine; AST, asparagine aminotransferase; DDAH, dimethylarginine dimethylaminohydrolase; ELISA, enzyme-linked immunosorbent assay; eNOS, endothelial nitric oxide synthase; HPLC, high-performance liquid chromatography; HSP70, 70 kDa heat shock protein; iNOS, inducible nitric oxide synthase; IR, ischemia/reperfusion; L-NMMA, NG-monomethyl-L-arginine; MIP-2, macrophage inﬂammatory protein 2; NADPH, nicotinamide adenine dinucleotide phosphate; NF-kappa B, nuclear factor kappa B; NO, nitric oxide; PRMTs, protein arginine methyltransferases; ROS, reactive oxygen species; SDMA, symmetric dimethylarginine; TNFα, tumor necrosis factor α. ⁎ Corresponding author. E-mail address: [email protected] (T. Sozański). 0531-5565/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.exger.2013.11.004 Ischemia/reperfusion (IR) is considered to be the main cause of structural and functional damage of the liver during surgery procedures such as liver transplantation and hepatic resection. IR is a double-stage process: initially it is caused by ischemia that is later aggravated by the reperfusion of the liver. Reperfusion injury involves an early acute phase (3–6 h after reperfusion), associated with Kupffer cell activation and generation of free radicals, nitric oxide (NO) production, and T-lymphocyte activation, followed by a subacute phase (18–24 h after reperfusion), characterized by a neutrophil inﬁltration leading to continuous oxidants, 46 M. Trocha et al. / Experimental Gerontology 50 (2014) 45–51 cytokines, and chemokine production (Fan et al., 1999; Hines et al., 2005). Endothelial nitric oxide synthase (eNOS) is responsible for the basal production of NO (Shah et al., 1997), but a higher level of NO produced by inducible nitric oxide synthase (iNOS) can promote an IR injury. NO is involved in the inﬂammatory process and modulates the metabolism of reactive oxygen species (ROS) (Fan et al., 1999; Peralta et al., 2001). NOS inhibitors such as asymmetric dimethylarginine (ADMA) may inﬂuence the NO level in the liver. ADMA released from the ischemic organ during the reperfusion phase competes with arginine for the binding site in the active center of NOS (Martin-Sanz et al., 2003; Vallance and Leiper, 2004). Dimethylarginine dimethylaminohydrolase (DDAH) – the enzyme that metabolizes ADMA – may inﬂuence the concentration of this compound (Leiper et al., 2002). A correlation between the concentration of methylarginine derivatives and the liver function and survival after a liver transplantation was observed (Martin-Sanz et al., 2003). The aging process is a multifactorial biological phenomenon characterized by the loss of adaptive responses to conditions of physiological stress, resulting in an increased susceptibility to diseases and death (Sohal et al., 2002). In recent times, the use of organs from donors older than 50 years has increased (Alkofer et al., 2006; Foster et al., 2007). However, liver function appears to be well maintained in old age (Fu and Nair, 1998), though numerous changes in hepatic structure and function have been described as related to age and there is an increase in the risk of organ failure after IR (Feng et al., 2006). It has also been shown that aged livers are more susceptible to IR, which results in an increased morbidity and mortality after vascular clamping (Clavien et al., 2003; Le Couteur et al., 1994). Some reports indicate that aged livers have an excellent capacity to regenerate after both partial hepatectomy and transplantation (Anantharaju et al., 2002; Schmucker, 1998), but a growing body of evidence suggests poor long-term survival after a liver transplantation (Collins et al., 2000). Distinct responses of old and young livers to IR were reported in certain papers (Abe et al., 2009; Kireev et al., 2012; Okaya et al., 2005; Trocha et al., 2007), but our knowledge is still incomplete. If damage extent in a liver preserved for transplantation depends largely on ADMA/DDAH/NO pathway it is reasonable to ask how the aging-process inﬂuences some parameters of that pathway under IR conditions. The aim of the study was to evaluate age-related differences in selected parameters, determining the NO level in young and mature livers subjected or not subjected to IR. 2. Materials and methods 2.1. Animals A study was carried out on Wistar male rats obtained from the Animal Laboratory of the Department of Pathological Anatomy, Wrocław Medical University. The animals were housed individually in chambers with a 12:12 h light–dark cycle with the temperature maintained at 21– 23 °C. Before the experiment, the animals had free access to standard food and water. The experiment was performed in accordance with NIH Guide for the Care and Use of Laboratory Animals and was approved by the Local Ethical Committee on Animal Research of the Institute of Immunology and Experimental Therapy, Polish Academy of Sciences in Wrocław. 2.2. Chemicals Heparin (Heparinum WZF—amp. 25,000 U/5 ml, Polfa Warszawa, Poland), ketamine hydrochloride (Bioketan, Vetoquinol Biowet, Poland), medetomidine hydrochloride (Domitor, amp. 1 mg/ml, Orion Pharma, Finland), 0.9% sodium chloride solution (Polpharma S.A., Poland), and Ringer solution (Polfa Lublin S.A., Poland) were used in this study. 2.3. Experimental design After adaptation, the rats were randomly divided into four groups: two groups (C-2 and IR-2) of young rats (2–4 months old) and another two groups (C-12 and IR-12) of older rats (12–14 months old). Rats belonging to the C-2 (n = 10) and the C-12 (n = 9) group were not subjected to IR conditions, and rats from the IR-2 (n = 9) and the IR-12 (n = 9) group were subjected to 60 min of partial ischemia followed by 4 h of global reperfusion. 2.4. Preparation of the liver IR injury model Rats were weighed and anesthetized with intramuscular injection of ketamine (7 mg/kg) with medetomidine (0.1 mg/kg) and underwent midline laparotomy. In the IR-2 and the IR-12 groups a 70% liver ischemia (left lateral and median lobes) was achieved by occlusion of branches of the portal vein and the hepatic artery using a microvascular clip. Rats were administered heparin (200 U/kg) to prevent blood coagulation. After 60 min of ischemia, the clip was removed to allow reperfusion for 4 h. The abdomen was subsequently closed and the rats were observed during reperfusion. At 15, 120 and 240 min of reperfusion blood samples were collected from the tail vein to determine the level of arginine (Arg), and its derivatives: ADMA and symmetric dimethylarginine (SDMA). At the end of reperfusion further blood samples were obtained to estimate activity of ALT and AST. When the experiment was terminated, the livers were weighted and ischemic lobes were isolated. In the C-2 and the C-12 groups the animals were anesthetized in the same way as those in the ischemic groups. Following this, the midline laparotomy branches of the portal vein and the hepatic artery were isolated but not occluded. After 60 min, the abdomen was closed and the rats were being observed for 4 h. Blood samples were obtained at the same points of time as in the case of the ischemic groups. After 4 h, the animals were terminated and the left lateral and median lobes of the livers were isolated to be compared with corresponding lobes obtained from the ischemic livers. 2.5. Blood enzyme, arginine and its derivatives, tissue nitric oxide synthase and DDAH analyses In liver homogenates DDAH activity was estimated using the colorimetric method (spectrophotometer MARCEL S350 PRO, Marcel sp. Z o. o., Poland) and was expressed per gram of protein. The method is based on the L-citrulline production rate. For that purpose, liver homogenate was mixed with a phosphate buffer, pH = 6.5. 1 mM ADMA was added to each sample and samples were incubated at 37 °C for 45 min. After the reaction was stopped by the addition of 4% sulfosalicylic acid, samples were centrifuged. Oxime (diacetic monooxime (0.08% w/v) in 5% acetic acid) mixed with antipyrine (antipyrine (0.5% w/v) in 50% sulfuric acid) was added to the samples at the next stage. Following 110 min of incubation at 60 °C and 10 min of cooling on ice, L-citrulline was determined at 466 nm wavelength. Obtained values were subtracted from the corresponding values in blind samples (without ADMA). Standard values were prepared as aliquots of L-citrulline. DDAH activity was presented as μm of L-citrulline/g of protein/min at 37 °C. The protein level for iNOS was determined in liver homogenates supernatants using commercially available enzyme-linked immunosorbent assay (ELISA) kit (USCN, Life Science Inc., UK). All samples and standards were performed in duplicates. The results were expressed in ng/ml. Arginine, ADMA, and SDMA concentrations were measured simultaneously by high-performance liquid chromatography (HPLC) with ﬂuorescence detection (Boger et al., 1998; Parker et al., 2003). Plasma samples and standards were extracted on a solid-phase extraction cartridge with SCX 50 columns (Varian, Palo Alto, USA). Analytes were M. Trocha et al. / Experimental Gerontology 50 (2014) 45–51 derivatized with o-phthaldialdehyde and separated by isocratic reversed-phase chromatography on a Symmetry C18 column (150 × 4.6 mm, 5 μm particle size; Waters Corp., Milford, MA, USA). Potassium phosphate buffer (50 mM, pH 6.6) containing 12% v/v acetonitrile was used as the mobile phase at a ﬂow rate of 1.1 ml/min and a column temperature of 35 °C. Fluorescence detection was performed at the excitation and emission wavelengths of 340 and 450 nm, respectively. The serum activities of ALT and AST and the concentration of protein in the homogenates were assayed with commercial methods in a certiﬁed laboratory. Total protein concentrations in supernatants of homogenates were assayed with commercial methods in a certiﬁed laboratory on the Dimension RxL-Max apparatus on Flex kit. Brieﬂy, cuprum-cation interacts with a peptide bond in protein in an alkaline solution; the amount of Cu(II) complex with blue color, proportional to protein concentration, is measured using bichromatic technique of ﬁnal point assessment. 2.6. Statistical analysis Data were expressed as the mean values ± SD. Statistical analysis of the effect of the age and IR on iNOS level, as well as DDAH and aminotransferases activity, was performed using a two-way analysis of variance (ANOVA). Statistical analysis of the effect of age and the time of reperfusion on Arg, ADMA, and SDMA levels and Arg/ADMA (A/A) ratio was performed using MANOVA with repetition. Speciﬁc comparisons were made using contrast analysis. The hypotheses were considered positively veriﬁed if p b 0.05. 3. Results 3.1. ADMA, SDMA, Arg, and A/A ratio The concentration of ADMA measured before IR was comparable in all groups of rats regardless of age. At 15 min of reperfusion in the IR2 group the increase in ADMA concentration was observed and the difference between that group and the C-2 group was signiﬁcant (IR-2 vs. C-2, p b 0.05). Otherwise, the concentration of ADMA was signiﬁcantly decreased in the ischemic group of the older rats (IR-12 vs. C-12, p b 0.05). Those changes in the ischemic groups were the reason for the signiﬁcant difference between young and older ischemic groups (IR-2 vs. IR-12, p b 0.001). At 120 min of reperfusion the concentration of ADMA in the IR-12 group slightly increased and achieved comparable level as in non-ischemic groups. At this point in time, the concentration of ADMA in the IR-2 group was still the highest of all the groups (IR-2 vs C-2, p b 0.05; IR-2 vs. IR-12 and C-12, p b 0.005 in both comparisons). In 240 min of reperfusion, the level of ADMA decreased in all the groups. However, it was still signiﬁcantly higher in the IR-2 group than in group of older ischemic rats (IR-2 vs. IR-12 and C-12, p b 0.05 in both comparisons). No signiﬁcant differences between the C-2 and the C-12 groups in all time points were observed (C-2 vs. C-12, p = NS) (Fig. 1A). Concentrations of SDMA were highest in the IR-2 group at all points of reperfusion. At the end of reperfusion it was signiﬁcantly higher in that group compared to the C-12 group (IR-2 vs. C-12, p b 0.05) and was on the border of signiﬁcance compared to IR-12 (IR-2 vs. IR-12, p = 0.05) (Fig. 1B). When analyzing particular time points of reperfusion it was observed that values of A/A ratio changed in the same way as Arg levels. The initial values of both parameters were signiﬁcantly higher in the young groups than in older ones (C-2 vs. C-12, p b 0.005 for Arg, C-2 vs. C-12, p b 0.05 for A/A ratio as well IR-2 vs. IR-12, p b 0.001 for both parameters). After the IR period the values of those parameters decreased at the ﬁrst 15 min and were signiﬁcantly lower from that in the non-ischemic group (IR-2 vs. C-2, p b 0.001 for both parameters). At all points of time (15, 120 and 240 min) the values of Arg level and A/A 47 ratio were highest in the C-2 group and the differences between that group and all the others were signiﬁcant (C-2 vs. IR-2 and IR-12, p b 0.001 for both parameters at all points of time; C-2 vs. C-12, p b 0.05 for Arg at 15 min and p b 0.01 for Arg at 120 and 240 min, C-2 vs. C-12 p b 0.05 for A/A ratio at all points of time). Differences between both groups of older rats were also signiﬁcant. In the non-ischemic groups of rats Arg level and A/A ratio were higher than in the ischemic ones at all points of time (C-12 vs. IR-12, p b 0.05 for both parameters at 15 min; p = 0.05 for Arg at 120 min, p b 0.01 for Arg at 240 min, and p = 0.07 for A/A ratio at 120 and 240 min). At the end of reperfusion the values of Arg level and A/A ratio were still highest in the C-2 group. In the groups of older rats the values of those parameters were higher in the nonischemic group than in the ischemic one (C-12 vs. IR-12, p b 0.01, for Arg, and p = 0.07 for A/A ratio) (Fig. 1C, D). 3.2. Biochemical analyses 3.2.1. iNOS In the C-2 group, iNOS protein concentration was higher than in the C-12 group and the difference between those groups was on the border of signiﬁcance (C-2 vs. C-12, p = 0.056). The increase in iNOS protein level was observed in the IR-2 group compared to the C-2 group (IR-2 vs. C-2, p b 0.005), which was not revealed in the groups of mature animals (C-12 vs. IR-12, p = NS). Therefore, there was a signiﬁcant difference between young and older rats subjected to IR (IR-2 vs. IR-12, p b 0.001) (Fig. 2A). 3.2.2. DDAH activity Activity of DDAH was on a comparable level in the non-ischemic groups regardless of age. The decrease in DDAH activity was observed in the IR-2 compared to the C-2 group (IR-2 vs. C-2, p b 0.001), which was not revealed in the groups of older animals (C-12 vs. IR-12, p = NS). Therefore, there was a signiﬁcant difference between young and older rats subjected to IR (IR-12 vs. IR-2, p b 0.001) (Fig. 2B). 3.2.3. Aminotransferases activity Aminotransferases (ALT, AST) activity was higher in the groups of older rats (C-12, IR-12) than in the young ones (C-2, IR-2) but the differences between both non-ischemic groups (C-2 vs. C-12, p = NS) and ischemic groups (IR-2 vs. IR-12, p = NS) were not signiﬁcant. The activity of ALT was signiﬁcantly higher in the groups subjected to IR than in the non-ischemic groups regardless of age (IR-12 vs. C-12, p b 0.01 and IR-2 vs. C-2, p b 0.05). Similarly, the activity of AST was signiﬁcantly higher in the IR-12 than in the C-12 group (IR-12 vs. C-12, p b 0.05), and was on the border of signiﬁcance in the groups of young rats (IR-2 vs. C-2, p = 0.08) (Table 1). 4. Discussion The demand for liver transplantation is continually growing and exceeding organ availability. It has become a procedure of choice in the treatment of chronic and acute liver failure. In the USA, more than 3000 liver transplantations are performed annually (Habior et al., 2002). On the other hand, in today's aging societies, an increasing number of potential organ donors are found amongst the elderly, so the use of elderly donors (over 50 years) has signiﬁcantly increased (Alkofer et al., 2006; Foster et al., 2007). For our experiment, male Wistar rats of two different ages, young (2–4 months) and older (12–14 months), were chosen, which corresponds approximately to a 10- to 15-yearold age range and an over 50-years-old in humans, respectively (Feng et al., 2006). A number of distinct age-related alterations have been identiﬁed in the hepatic response to IR, including amongst others, a reduced production of antioxidants and a more evident inﬂammatory response (increased expression of pro-inﬂammatory genes and cytokines and decreased mRNA expression of antiinﬂammatory cytokines), a lower expression of the cytoprotective protein (HSP70), increased 48 M. Trocha et al. / Experimental Gerontology 50 (2014) 45–51 Fig. 1. Inﬂuence of IR and aging process on levels of ADMA (A), SDMA (B), Arg (C) and Arg/ADMA ratio (D). Values are presented as the mean ± SD. C-2—group of young rats (2–4 months old) non-subjected to IR, IR-2—group of young rats (2–4 months old) subjected to IR, C-12—group of older rats (12–14 months old) non-subjected to IR, IR-12—group of older rats (12–14 months old) subjected to IR. Speciﬁc comparisons: #p b 0.05, ###p b 0.005 (compared to C-2); *p b 0.05, **p b 0.01 and ***p b 0.005 (compared to IR-2), Δp b 0.05, ΔΔΔp b 0.005 (compared to C-12). tissue injuries (ALT and AST serum level, neutrophil inﬁltration and function), as well as a decrease in the liver's regenerative capacity (Kelly et al., 2011; Massip-Salcedo et al., 2007). However, our knowledge of the mechanisms of such a greater susceptibility to IR conditions in livers from older donors is still incomplete. So far, too little data concerning the impact of the aging process on ADMA–DDAH–NO pathway in a liver subjected to IR has been reported. Therefore, our experiment was designed to assess how the aging process affects certain parameters determining nitric oxide level during IR. We decided to examine these parameters in an earlier period of reperfusion because the ﬁrst few hours of reperfusion seem to be of greatest importance for this organ. The results of this study showed important differences in the response of the two age groups of rats to IR conditions. The most relevant conclusions are presented below: 1) The concentration of ADMA was different depending on the age, which was seen particularly in the ﬁrst 2 h of reperfusion. It decreased and increased signiﬁcantly in the ischemic groups of mature and young animals, respectively; 2) Only in the young ischemic group was the increase in SDMA concentration observed; 3) The initial values of Arg level and A/A ratio were signiﬁcantly higher in the groups of young rats compared to the mature animals, but under IR a decrease in those parameters was observed regardless of age; 4) iNOS protein concentration was signiﬁcantly higher in the young than in the older group; 5) Only in the ischemic group of young rats did DDAH activity decrease. NO, through its vasodilative effect, may maintain perfusion and prevent endothelial injury (Kobayashi et al., 1995). It can also accept other electrons as a free-radical scavenger (Ignarro, 1989). However, an excessive production of NO, mainly by iNOS, is cytotoxic and may react with superoxide to form toxic peroxinitrite (Fan et al., 1999). iNOS was shown to aggravate liver injury. Up-regulation of this isoform plays a signiﬁcant role in the inﬂammatory process in the liver and that inhibition of iNOS reduces liver injury (Fan et al., 1999; Hierholzer et al., 1998). But it was also shown that (Rivera-Chavez et al., 2001) the use of selective inhibitor of iNOS did not provide any signiﬁcant protection in the liver function and survival. In our experiment the increase in iNOS protein concentration after reperfusion was observed only in the livers derived from the young animals. In mature organs, which are considered to be more susceptible to IR, iNOS protein concentration was on a similar level as the group not subjected to IR. The role of iNOS in the early phase of reperfusion remains unclear and more work is needed to elucidate its role in a liver under IR. Interestingly, the results obtained in an experimental model of an isolated perfused liver indicated that during reperfusion livers obtained from mature animals generated a lower amount of free radicals (Gasbarrini et al., 1998) and exerted delayed activation of NF-kappa B in response to TNF alpha, as well as no production of MIP-2 (Okaya et al., 2005) compared to livers M. Trocha et al. / Experimental Gerontology 50 (2014) 45–51 Fig. 2. Inﬂuence of IR and aging process on iNOS protein concentration (A) and DDAH activity (B). Values are presented as the mean ± SD. C-2—group of young rats (2–4 months old) non-subjected to IR, IR-2—group of young rats (2–4 months old) subjected to IR, C-12—group of older rats (12–14 months old) non-subjected to IR, IR-12—group of older rats (12–14 months old) subjected to IR. Speciﬁc comparisons: ###p b 0.005 (compared to IR-2). derived from young animals. NF-kappa B is a primary transcriptional regulator of various proinﬂammatory mediators, such as iNOS. The described differences could be explained by lower Kuppfer cell activity and the reduction of the liver blood ﬂow. In our work, changes in iNOS protein concentration should be analyzed in relation to other parameters. It is worth noting that in the young rats DDAH activity decreased and ADMA concentration increased during the ﬁrst 2-hours of reperfusion. Therefore increased iNOS level is necessary to maintain the proper level of NO. Finally, it is possible that changes in the synthase protein concentrations do not directly reﬂect the increased or decreased enzyme activity. Hines suggested that changes in NO synthesis during reperfusion may occur as a result in changes of enzyme function, not its concentration (Hines et al., 2005). NO is synthesized mainly from Arg by NOS and its level is determined by various factors and diseases (Lhuillier et al., 2003). Low NO levels after ischemia may be related to a low intracellular level of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) and tetrahydropterin (eNOS cofactors), limited oxygen support, increased Table 1 Values of ALT and AST activity after 60 min of ischemia and 240 min of reperfusion (IR-2, IR-12 groups) or without IR (C-2, C-12 groups). Values are presented as the mean ± SD. Speciﬁc comparisons: #p b 0.05, (compared to C-2); Δp b 0.05, ΔΔΔp b 0.005 (compared to C-12). Groups C-2 (n = 10) IR-2 (n = 9) C-12(n = 9) IR-12 (n = 9) ALT (U/l) AST (U/l) Mean ±SD Mean ±SD 107.40 867.78# 148.56 1344.33ΔΔΔ 45.59 634.27 53.51 1516.11 496.00 1307.56 607.00 1711.33Δ 100.72 677.90 169.62 1890.03 49 activity of argininase that removes Arg required for NO synthesis, and ﬁnally to increased release of NOS inhibitors from the ischemic organ during reperfusion (Martin-Sanz et al., 2003). It was reported that during an anhepatic phase and just after surgery, the level of ADMA – inhibitor of NOS – was elevated but within 1 h after liver transplantation the ADMA level was signiﬁcantly reduced, which was associated with the improvement in organ function (Mookerjee et al., 2007). In our work, no important changes of initial ADMA in either the young or the mature animals were observed. But, under IR conditions, during the ﬁrst 15 min of reperfusion, ADMA concentration was increased in the young ischemic group and decreased in the old ischemic group. After 240 min of reperfusion, the concentration of ADMA was more parallel between the groups but it was still higher in the ischemic group of young livers. The extensive production of ADMA could be responsible for the deterioration of the balance between NO and endothelin and consequent vasoconstriction which affects the liver function under IR (Laleman et al., 2005). Such a difference in the concentration of ADMA could also appear in response to a more intensive iNOS-dependent NO synthesis in the group of young rats. Similarly, in other reports ADMA is suggested to play the role as an inhibitor in the overproduction of NO, mainly by the regulation of iNOS activity (Kang et al., 1999; Trocha et al., 2010a, 2010b; Ueda et al., 2003). During reperfusion, a large amount of NO derived from cytokine-induced iNOS can react with superoxide to form peroxinitrite and augment cell injury (Buttery et al., 1996). An increased level of ADMA could prevent young livers from an extensive production of NO. SDMA, Arg and A/A ratio may also inﬂuence NO bioavailability. The correlation between SDMA and the serum creatinine level in both liver donors and recipients suggest that SDMA may have a diagnostic value in the progression of chronic kidney damage (Wnuk et al., 2012). Nevertheless, the role of SDMA in an injured liver is not fully understood. In our experiment the concentrations of SDMA were highest in the ischemic group of young rats, and similarly to ADMA level. It is difﬁcult to explain this fully on the basis of currently available data. The relationship between ADMA and Arg level is very important under pathological conditions, such as IR and, therefore, both the ADMA level and the A/A ratio need to be assessed. Arg is the main substrate for NO synthase, and a deﬁciency of this compound causes a drop in NO synthesis (Furchgott, 1996). It seems that various effects of Arg depend on an initial ADMA level and administration of Arg is justiﬁed in patients with low level of A/A ratio. It was reported that Arg administration results in normalization of the endothelium-dependent relaxation in patients with hypercholesterolemia (Boger et al., 1998) or in patients with chronic heart failure. But such effect was not observed in patients with low ADMA levels (Hornig et al., 1998). In our work, we have demonstrated a few important issues related to the Arg level and A/A ratio. First, the initial values of both parameters were signiﬁcantly higher in the young groups than in the older ones which may indicate that hepatic protection depends on Arg level and is more evident in the young animals. Second, the values of the A/A ratio changed in the same way as the Arg level during the whole reperfusion. Third, values of both parameters decreased in the ﬁrst 15 min of reperfusion and were signiﬁcantly lower than that in the non-ischemic group in both the young and the mature animals. Those results are similar to other reports in which an increase in ADMA level and a decrease in Arg concentration in various pathological states have been reported (Dimitrow et al., 2007). The inﬂuence of IR injury was observed in our previous experiments (Trocha et al., 2010a, 2010b, 2013) also in which the highest decrease of Arg level and A/A ratio was observed during the ﬁrst 90-minutes of the experiment. But, for the ﬁrst time age related differences in Arg level and A/A ratio in response to IR were shown. The decrease in Arg level and A/A ratio may suggest that the protection of the liver during the early period of reperfusion is similarly reduced regardless of age even though there were differences in the concentrations of ADMA and SDMA. It is worth noting that at the end of reperfusion, ADMA as well SDMA and Arg level were more parallel amongst all the groups indicating that 50 M. Trocha et al. / Experimental Gerontology 50 (2014) 45–51 the monitoring of changes in those parameters during the experiment is more valuable than a single measurement at the end of reperfusion. DDAH metabolizes NG-monomethyl-L-arginine (L-NMMA) or ADMA to citrulline and methylamine or dimethylamine, respectively (Leiper and Vallance, 1999; Tran et al., 2003; Vallance and Leiper, 2004). This reaction is present in both liver endothelial cells (Scalera et al., 2004) and hepatocytes (Mishima et al., 2004). Simultaneously with an altered liver function, ADMA concentration is usually increased mainly due to an inhibited degradation (Nijveldt et al., 2003). Cysteine, located in the active site of this enzyme, is sensitive to oxidation or nitrosation with subsequent loss of enzyme activity (Leiper et al., 2002). A growing body of evidence suggests that in hypertension, hypercholesterolemia, hyperglycemia, and hyperhomocysteinemia, oxidative stress is the main factor affecting DDAH activity leading to the increased ADMA concentration (Boger et al., 1998; Lin et al., 2002; Xiong et al., 2003). However, there are papers that do not conﬁrm such effect of the oxidative stress (Chobanyan et al., 2007). The results of our study have shown, for the ﬁrst time, that activity of DDAH was on a comparable level in the nonischemic groups regardless of age, but under IR in the young animals the decrease in DDAH activity was observed. It is difﬁcult to explain why we did not observe the same changes in the group of young and mature rats. It may suggest that DDAH activity was decreased in the young animals in response to a higher level of iNOS and was there to maintain NO level. Also in another report it was investigated that sixfold increases in NO produced by iNOS after cytokine treatment of endothelial cells are sufﬁcient to inhibit DDAH activity, probably followed by an increased ADMA concentration (Leiper et al., 2002). One of the limitations of this work is the use of a model of partial liver ischemia. The principal disadvantage is that during the induced ischemia of median and left lateral lobe, increased blood ﬂow in the rest of the liver occurred (sinusoidal overproduction). This affects the liver's function and regeneration. Because of the collateral blood supply, the exchange of oxygen from the perfused to the ischemic part of the liver can take place. Therefore, such differences in examined parameters were not as evident as in transplantation surgery. It is worth noting that livers derived from the non-ischemic groups were also subject to a gentle in situ organ manipulation (Schemmer et al., 1999). In this way we tried to exclude differences that might have appeared as a result of surgery procedure. The ALT and AST activity in our work was higher in the group of mature animals than in the young ones in both physiological and IR conditions but, contrary to other results (Okaya et al., 2005; Trocha et al., 2007), those changes were not important. Only the inﬂuence of IR was observed because after 4 h of reperfusion the activity of those enzymes increased statistically regardless of age. Summing up, in our study we have demonstrated, for the ﬁrst time, that the inﬂuence of IR on ADMA–DDAH–NO pathway in rat livers is strongly dependent on age. The elevation of ADMA, SDMA and iNOS protein concentration, the reduction of Arg level and A/A ratio, as well as the DDAH activity, were observed only in young animals. It is unclear whether the changes in DDAH are the result or the cause of the increase in ADMA level. Another question is why in the group of older rats such changes were not observed. One possible explanation is the reduction of blood ﬂow or the impaired function of Kuppfer cells in this age (Gasbarrini et al., 1998). Another important ﬁnding is the signiﬁcantly higher level of Arg and A/A ratio in the young compared with the mature animals in physiological conditions, which conﬁrms earlier ﬁndings that the livers from aged animals are more susceptible to possible toxic factors. In order to understand these results better our data should be supplemented in the future by the assessment of additional parameters of NO metabolism such as the activity of protein arginine methyltransferases (PRMTs)—enzymes responsible for protein methylation or other isoforms of NOS. Additionally, our experiment focused only on the earlier phase of reperfusion and in the future such observations should also be provided after a prolonged time of reperfusion. However, the results of this work may just indicate that the changes in particular parameters determining the NO level are dependent on age and may be the reason for the variable response of the liver to such pathological conditions as those observed under IR. 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