Brain & Development 30 (2008) 391–395 www.elsevier.com/locate/braindev
Isoprostanes in dystrophinopathy: Evidence of increased oxidative stress Salvatore Grosso a, Seraﬁna Perrone a, Mariangela Longini a, Carlo Bruno b, Claudio Minetti b, Diego Gazzolo c, Paolo Balestri a, Giuseppe Buonocore a,* a b
Department of Pediatrics, Obstetrics and Reproductive Medicine, Viale Mario Bracci 36, 53100 Siena, Italy Department of Pediatrics and Neuroscience, G. Gaslini Children’s Hospital, University Hospital, Genoa, Italy c Department of Maternal, Fetal, and Neonatal Health, G. Garibaldi Hospital, Catania, Italy Received 18 May 2007; received in revised form 14 November 2007; accepted 22 November 2007
Abstract Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are degenerative disorders of muscle. Although the mechanisms underlying muscle degeneration are still uncertain, oxidative-damage has been proposed to play a key role. Isoprostanes are markers of free radical-catalyzed lipid peroxidation; the aim of our study was to evaluate plasma isoprostane levels in group of patients aﬀected by Duchenne and Becker muscular dystrophies. PF2-isoprostane levels were measured by colorimetric enzyme immunoassay in the plasma of 17 patients with DMD and 24 with BMD. When compared to a group of healthy controls, aﬀected patients showed signiﬁcantly higher plasma levels of isoprostanes (p = 0.001). When patients were stratiﬁed according to the clinical diagnosis, isoprostane levels were not statistically diﬀerent between DMD and BMD patients. In conclusion whether the condition of oxidative stress found in plasma depends on the degenerative process occurring in muscles or on diﬀerent mechanisms, such as the release of myoglobin in the blood, should be ascertained. However, our study conﬁrms that oxidative stress ﬁndings in DMD/BMD patients are eﬀectively present at the plasma levels. The condition of oxidative stress might act as an adjunctive cause of extra-muscular cell damage to which these patients are exposed for their entire life. Ó 2007 Elsevier B.V. All rights reserved. Keywords: Oxidative stress; Duchenne muscular dystrophy; Becker muscular dystrophy; Muscle disorders
1. Introduction Duchenne muscular dystrophy (DMD) and the milder clinical form Becker muscular dystrophy (BMD), are fatal degenerative disorders of muscle resulting from mutations in the gene coding for dystrophin. That protein serves to link actin ﬁlaments within the muscle cell to a complex of glycoproteins in the sarcolemmal membrane . The absence or an abnormal dystrophin is thought to result in muscle membrane instability which becomes susceptible to contraction*
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induced cellular damage . However, the exact mechanisms through which the absence or an abnormal dystrophin result in muscle degeneration are still uncertain [3,4]. Previous experimental data suggested that oxidative-damage may play a key role in the pathogenesis of muscle degeneration which characterizes DMD/BMD. Indeed, it has been found that the neuronal isoform of nitric oxide (nNO) synthase, is associated with dystrophin–glycoprotein complex (DGC) in the sarcolemma of fast-twitch muscle ﬁbers . The impaired NO production in the muscle ﬁbers of DMD/BMD patients make them to be unprotected against the damaging actions of the reactive oxygen species (ROS) [2,5,6].
S. Grosso et al. / Brain & Development 30 (2008) 391–395
Globally, ROS may be generated by several sources including phagocyte activation, catecholamine metabolism, mitochondrial dysfunction, arachidonic acid cascade, and Fenton reaction driven by non protein bound iron (NPBI). Direct markers of oxidative stress include isoprostanes and malondialdehyde, the lipids, and protein carbonyl groups [7–9]. Isoprostanes are prostaglandin (PG)-like substances that are produced in vivo independently of cyclooxygenase (COX) enzymes, primarily by free radicalinduced peroxidation of arachidonic acid . The formation of PG-like compounds during auto-oxidation of polyunsaturated fatty acids was ﬁrst reported in the mid-1970s, but isoprostanes were not discovered to be formed in vivo in humans until 1990 . F2-isoprostanes are a group of 64 compounds isomeric in structure to cyclooxygenase-derived PGF2a. Other products of the isoprostane pathway are also formed in vivo by rearrangement of labile PGH2-like isoprostane intermediates. These include E2- and D2-isoprostanes, cyclopentenone-A2- and J2-isoprostanes, and highly reactive acyclic-ketoaldehydes (isoketals) [8–10] (Fig. 1). The objective of the present investigation was therefore to determine whether there was evidence for increased plasma levels of isoprostanes as a markers of oxidative stress in a series of patients aﬀected by DMD/BMD.
2. Patients and methods 2.1. Patients Forty-one patients with age ranging from 3.5 years to 26 years were enrolled into the study. Seventeen patients were aﬀected by DMD (8.7 ± 4.3 years of age) and 24 by BMD (16.0 ± 9.6 years). In all patients, clinical diagnosis was conﬁrmed by molecular analysis as previously described. The group of patients underwent blood sample withdrawn. The control group consisted of 20 young healthy boys who were not active in sports or physical activity (age = 14.0 ± 7.0 years). None of the participants were taking creatine monohydrate supplements or speciﬁc antioxidant supplements at the time of collection. Completed written informed consent forms was obtained from all the subjects, or their parents, before participation in the study. 2.2. Isoprostanes F2-IP assessment were performed on plasma. Butylated hydroxytoluene (BHT) was added to prevent oxidation during processing. Samples were centrifuged at 1000 rpm, and the supernatant was stored at 80°C. F2-IP were determined in a single matrix. Ethanol was added to remove precipitated protein and acetate buﬀer added up to pH 4.0 for 15-F2t-IsoP determina-
Arachidonic acid esterified to phospholipids
Free radical attack Arachidonoyl radicals
Peroxidation Isolevuglandins H2-isoprostanes endoperoxides
Fig. 1. Isoprostane pathway of free radical-induced oxidation of arachidonic acid.
S. Grosso et al. / Brain & Development 30 (2008) 391–395
tion. Samples were puriﬁed of the metabolite with C18 and silica Sep-Paks cartridges (Waters Co., Milford, MA) before a colorimetric enzyme immunoassay (Cayman Chemical, Ann Arbor, MI). The antibody was highly speciﬁc for 15-F2t-IsoP (8-Iso PGF2). The range of the standard curve was from 3.9 to 500 pg/ml and the detection limit was 2 pg/ml. Serial sample dilutions were parallel to the standard curve. The intra-assay coeﬃcient of variation of F2-IP was 62%. 2.3. Statistical evaluation Statistical analysis was performed using a one-way, between-group analysis of variance with signiﬁcance set at p < .05. Data were expressed as means ± standard deviation. 3. Results Patients with dystrophinopathy showed higher plasma levels of isoprostanes as compared to the controls (p < 0.001). No diﬀerences there were in isoprostanes plasma levels between the DMD patients and those aﬀected by BMD (p = 0.1) (Fig. 2). 4. Discussion ROS (particularly hydroxyl and superoxide) are ubiquitously produced during normal aerobic cellular metabolism, with the possibility of initiating damage to lipids, protein, and nucleic acids . A protective system against ROS actions is represented by a variety of endogenous ROS scavenging compounds, proteins, and enzymes [3,11,12]. The possible role of ROS in the pathogenetic mechanisms of muscle degeneration in
Fig. 2. The panel shows the means ± SD serum isoprostane levels (pg/ ml) in patients and healthy controls. Isoprostane levels were signiﬁcantly increased when compared to the control group (p = 0.001). By contrast, no signiﬁcant diﬀerences there were in isoprostanes serum levels between patients with Duchenne dystrophy and those aﬀected by Becker dystrophy (p = 0.1).
DMD/BMD, has been pointed out following the observation of an induced-muscular dystrophy process in animals submitted to nutritional vitamin E and selenium deprivation, which are well known antioxidant agents . Subsequently, several observations lent support to the hypothesis that one of the protective role of the DGC in the cell may be the prevention of oxidative injury by regulating cellular antioxidant defenses . Haycock et al.  demonstrated that the total cellular proteins present in DMD patients are quantitatively more oxidized than in normal muscle. In fact, carbonyl protein levels in the quadriceps femoris muscle were found to be 211% higher than normal. Interestingly, transgenic mice over-expressing copper/zinc (Cu/Zn) superoxide dismutase show high serum levels of CK and muscular histologic changes similar to those observed in typical muscular dystrophy. These ﬁndings conﬁrm that oxidative-damage plays an early role in the pathogenetic process of muscular dystrophies . In this context, it has been established that the susceptibility of dystrophin protein to free radicals precedes cellular necrosis . Disatnik et al.  observed a positive relationship between the type of dystrophin gene mutation and the susceptibility of muscle cell to oxidativedamage. In fact, muscle cells from mdx-transgenic strains expressing no dystrophin had higher susceptibility when compared with those from mdx-transgenic strains expressing full-length or truncated forms of dystrophin. However, recent investigations suggested that loss of NOS protection, alone, may not totally explain muscle ﬁber degeneration since mice lacking neuronal NOS do not develop a muscular dystrophy. Indeed, Kameya et al.  observed that knock-out mice for a-1-synotrophin, a dystrophin associated protein, did not show muscle degeneration. Additional biochemical changes, therefore, may be involved in the oxidative-damage leading muscle cell to degeneration. According to Rando et al. , pathogenetic defects in the DGC mainly have two biochemical consequences: an impaired nNO production, which determines a scarce protection of muscle cells against ischemia, and an increased cellular susceptibility to metabolic stress. This possible pathogenetic model has been called the ‘‘two-hit’’ hypothesis. The ‘‘two-hit’’ hypothesis may well explain many of the complex spatial and temporal variations in the disease expression that characterize the muscular dystrophy, such as the pre-necrotic stage of the disease, muscle cell necrosis, and the selective muscle involvement . The possible pathogenetic role of oxidative stress has also been pointed out by studies performed in patients aﬀected by some neurodegenerative conditions [18–20] and neuromuscular disorders such as mitochondrial defects and myotonic dystrophy type 1 . In particular, an increase in oxidative stress has also been found
S. Grosso et al. / Brain & Development 30 (2008) 391–395
in patients with DMD/BMD . However, the authors used a diﬀerent experimental approach to evaluate the oxidative stress. In fact the utilization of 8-hydroxy-2 0 deoxyguanosine (8-OHdG), which is a speciﬁc indicator of DNA damage, cannot evidence the total antioxidant status . In the present study, we studied oxidative stress condition at the plasma levels in a series of patients with DMD/BMD by evaluating plasma levels of isoprostanes. Isoprostanes represent the most direct and reliable markers of oxidative stress. They contain the F-type prostane ring are isomeric to PGF2a and are thus referred to as F2-IP. Isoprostanes are initially formed in situ on phospholipids, and rapidly released into the circulation. Since isoprostanes are less reactive than other lipid peroxidation products such as lipid hydroperoxides and aldehydes, they can be detected in biological ﬂuids [11,12]. Measurement of F2-IP is the most reliable approach to assess oxidative stress status in vivo, providing an important tool to explore the role of oxidative stress in the pathogenesis of human disease. Moreover, F2-IP and other products of the isoprostanes pathway exert potent biological actions both via receptor-dependent and independent mechanisms and therefore may be pathophysiological mediators of disease . High plasma and/or urinary levels of isoprostanes have been observed in several disorders such as diabetes, alcoholic liver disease, Alzheimer disease, and retinopathy of prematurity [9,23,24]. Elevated levels of isoprostanes have also been found in amniotic ﬂuid of fetal growth restriction pregnancies, resulting to be a reliable assessment of fetal oxidative stress [11,12]. We found increased levels of isoprostanes in patients aﬀected by dystrophinopathy in comparison with healthy subjects, conﬁrming therefore the increased oxidative stress in this population. Since the higher creatine kinase activity in plasma from patients with dystrophinopathy is indicative of muscle membrane dysfunction, and the expression of an increase in myocyte degeneration, we performed a correlation study between CK levels and isoprostanes. No statistically signiﬁcant results were found. Moreover, when the patients where stratiﬁed in accordance with the clinical diagnosis (DMD or BMD), no diﬀerences there were in the levels of plasma isoprostanes between the two groups. Due to the size of our series it was not possible to investigate on the relationship between isoprostane levels and the disability grade (ambulant or not) of patients. Further studies are needed to assess that issue. From a pathogenetic point of view, the relationship between muscle degeneration and plasmatic high levels of marker of oxidative stress, remains to be established. In fact, the increased markers of oxidative stress we found in the present series may of course be the direct expression of the many processes accompanying muscu-
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