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Reduced Antioxidant Defense and Increased Oxidative Stress in Spinal Cord Injured Patients

      Abstract

      Bastani NE, Kostovski E, Sakhi AK, Karlsen A, Carlsen MH, Hjeltnes N, Blomhoff R, Iversen PO. Reduced antioxidant defense and increased oxidative stress in spinal cord injured patients.

      Objective

      To determine the plasma and urine levels of antioxidants and oxidative stress biomarkers in subjects with spinal cord injury (SCI) the first year after injury.

      Design

      Descriptive 1-year follow-up study.

      Setting

      Rehabilitation and research center.

      Participants

      SCI subjects (n=37; age range, 18–70y) consecutively enrolled within the first month after injury. A healthy, able-bodied control group (n=346) was also included.

      Interventions

      Not applicable.

      Main Outcome Measures

      Blood and urine levels of antioxidants and oxidative stress biomarkers were measured at inclusion and after 3 and 12 months postinjury.

      Results

      One month after injury, the plasma antioxidants (total and oxidized glutathione and 6 different carotenoids and α-tocopherol) were reduced by 19% to 71% among the SCI subjects compared with the controls. The redox potential was reduced by 7% among the SCI subjects. The oxidative stress biomarker urinary 8-epi prostagladin F2α (PGF2α) increased to 161% in the SCI subjects compared with the controls. After 3 and 12 months, most of the antioxidant biomarkers were still significantly reduced compared with the controls, while urinary 8-epi PGF2α had increased to 208% compared with the controls.

      Conclusions

      The levels of antioxidants were significantly lower, while the marker of oxidative stress was higher in the SCI subjects compared with the controls. This observation demonstrates that SCI patients experience increased oxidative stress and reduced antioxidant defense the first year after injury. Our findings warrant intervention studies where SCI patients receive dietary antioxidant support as part of their rehabilitation.

      Key Words

      List of Abbreviations:

      AIS (American Spinal Injury Association Impairment Scale), Cys (cysteine), Cys/Gly (cysteinyl-glycine), GSH (glutathione), GSSG (oxidized glutathione), Hcy (homocysteine), HPLC (high-pressure liquid chromatography), PGF2α (prostagladin F2α), ROS (reactive oxygen species), SCI (spinal cord injury)
      IN THE NORDIC COUNTRIES, between 10 and 20 persons per million inhabitants annually suffer a traumatic spinal cord injury (SCI) that causes permanent and severe disability.
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      Survival after spinal cord injury in Finland.
      Patients with injury to 1 of the 8 cervical segments or the first thoracic segment of the spinal cord have tetraplegia, whereas those with paraplegia have lesions in 1 of the remaining caudal segments. Persons with incomplete lesions have spared sensory and/or motor function in the sacral segments and varying degrees of muscle function below the injury level.
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      Among those with incomplete SCI, motor and sensory function recovery occurs mostly within the first year after injury (early phase), although some also may have improvement in functions later (chronic phase).
      • Manella K.J.
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      • Field-Fote E.C.
      Restoration of walking function in an individual with chronic complete (AIS A) spinal cord injury.
      The most frequent neurologic deficits after traumatic SCI are incomplete tetraplegia, followed by complete paraplegia, incomplete paraplegia, and complete tetraplegia.
      An SCI is characterized by primary damage that is defined as the immediate effects of an injury to the spinal cord, most commonly caused by disruption, contusion, or compression. Then, secondary damage occurs in the following hours or days, which is caused by the body's local and general response to the trauma.
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      Recombinant human erythropoietin prevents motor neuron apoptosis in a rat model of cervical sub-acute spinal cord compression.
      Although the pathophysiologic mechanisms underlying the primary and secondary damages in SCI are incompletely understood, oxidative stress is believed to be involved.
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      Oxidative stress in spinal cord injury and antioxidant-based intervention.
      The generation of reactive oxygen species (ROS) (eg, free radicals) is a normative response to diseases or injuries. However, increased formation of the ROS can exceed the capacity of the antioxidant defense and may thus mediate oxidative damage and subsequently oxidative stress.
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      A number of endogenous and exogenous antioxidants are important for the antioxidant defense. The tripeptide glutathione (GSH) is an essential endogenous small molecular weight thiol present in plasma and in all cells in the body.
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      Glutathione, oxidative stress and neurodegeneration.
      The oxidation-reduction (redox) of plasma GSH, that is, the redox state of the GSH/oxidized glutathione (GSSG) couple, was estimated using the Nernst equation, as described by Schafer and Buettner.
      • Schafer F.Q.
      • Buettner G.R.
      Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple.
      A more negative value of GSH redox potential indicates more reduced GSH/GSSG redox state, and hence higher reducing capacity of GSH. An increase in GSSG in plasma decreases the GSH/GSSG ratio and results in more positive GSH redox potential, and hence lowers reducing capacity. In addition to the free reduced and oxidized forms of GSH in plasma, GSH is also bound to other thiol compounds (eg, cysteine) and proteins.
      • Iwasaki Y.
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      • et al.
      Chromatographic and mass spectrometric analysis of glutathione in biological samples.
      Therefore, the total GSH is also a measure of oxidative stress.
      Carotenoids are lipid soluble plant pigments with antioxidant activity. Among the estimated 750 naturally known carotenoids, lutein, zeaxanthin, β-kryptoxanthin, α-carotene, β-carotene, and lycopene are the most studied.
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      Aquatic animal carotenoids.
      Plasma carotenoids may also serve as biomarkers for fruit and vegetable intake.
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      Free radicals, antioxidants, and nutrition.
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      • Azzi A.
      The European perspective on vitamin E: current knowledge and future research.
      Vitamin E (α-, β-, γ-, and δ-tocopherols) is considered to be the major lipid-soluble antioxidant to protect cell membrane from oxidative damage.
      • Ayaori M.
      • Hisada T.
      • Suzukawa M.
      • et al.
      Plasma levels and redox status of ascorbic acid and levels of lipid peroxidation products in active and passive smokers.
      Isoprostanes are biologically active compounds formed by oxidation of polyunsaturated fatty acids. Urine isoprostane 8-epi prostagladin F2α (PGF2α) is often used as a biomarker for oxidative stress.
      • Morrow J.D.
      • Roberts L.J.
      The isoprostanes: their role as an index of oxidant stress status in human pulmonary disease.
      Despite the postulated role of oxidative stress in the development and progression of SCI, there is only limited information about oxidative stress and the status of the antioxidant system in SCI patients.
      • Jia Z.
      • Zhu H.
      • Li J.
      • Wang X.
      • Misra H.
      • Li Y.
      Oxidative stress in spinal cord injury and antioxidant-based intervention.
      Additionally, very little is known about temporal changes in oxidative stress after an acute trauma to the spinal cord. Moreover, the possible impact of oxidative stress on the severity of SCI remains largely unknown.
      In this study we have used a broad panel of antioxidant and oxidative stress biomarkers to determine antioxidant status in SCI patients 1, 3, and 12 months after acute trauma to the spinal cord in otherwise healthy subjects and compared the results with those from able-bodied controls.

      Methods

      Participants and Study Design

      We invited 85 adult (range, 18–70y) men and women with acute SCI hospitalized in Sunnaas Hospital (Nesoddtangen, Norway) from January 2007 to July 2009 to participate in the study. While 48 SCI subjects declined the invitation or did not fulfill the inclusion criteria, 37 otherwise healthy SCI subjects were included in the study. In particular, they did not suffer from any immunologic, inflammatory, or malignant disease, and they did not use any drugs regularly except for infrequent use of spasmolytics. Blood and urine samples were collected from the SCI subjects within the first month after injury, at discharge from Sunnaas Hospital (ie, after about 3mo), and as outpatients 1 year or more (chronic phase) after the trauma. Because of transfer to other hospitals or discharge, we were not able to obtain blood and urine samples from all patients. Samples were obtained from 37, 26, and 33 SCI patients after 1, 3, and 12 months, respectively.
      An SCI is classified by the American Spinal Injury Association Impairment Scale (AIS), which indicates the level of remaining sensory and/or motor function below the spinal level of injury. In short, AIS grade A injury indicates no preserved motor or sensory function, AIS grade B indicates preserved sensory function, but no preserved motor function, and AIS grades C and D injuries indicate preserved sensory function with varying degrees of loss of motor function.
      Additionally, we used data obtained from a control group consisting of 346 healthy individuals originally included in another study conducted at the University of Oslo.
      • Carlsen M.H.
      • Karlsen A.
      • Lillegaard I.T.
      • et al.
      Relative validity of fruit and vegetable intake estimated from an FFQ, using carotenoid and flavonoid biomarkers and the method of triads.
      • Sakhi A.K.
      • Bohn S.K.
      • Smeland S.
      • et al.
      Postradiotherapy plasma lutein, alpha-carotene, and beta-carotene are positively associated with survival in patients with head and neck squamous cell carcinoma.
      These 346 participants were recruited after response to an invitation letter sent to a random selection of adult citizens in Oslo and its surrounding area.
      The present study was conducted according to the Declaration of Helsinki guidelines. All procedures were approved by the Regional Committee for Medical and Health Research Ethics South, and all participants gave written informed consent at the time of inclusion.

      Blood and Urine Sampling

      Venous blood and spot urine were sampled from SCI patients in the morning after an overnight fast. Plasma and urine aliquots were snap-frozen in liquid nitrogen and stored at −80°C. Overnight, fasting plasma samples were available from all 346 control subjects, whereas 24-hour urine samples were available from 132 control subjects.

      Measurement of Plasma GSH/GSSG

      The determination of GSH and GSSG by high-pressure liquid chromatography (HPLC) has been detailed by Sakhi et al.
      • Sakhi A.K.
      • Blomhoff R.
      • Gundersen T.E.
      Simultaneous and trace determination of reduced and oxidized glutathione in minute plasma samples using dual mode fluorescence detection and column switching high performance liquid chromatography.
      The plasma obtained was prepared for analyses of reduced and oxidized forms of GSH, as described in Sakhi et al.
      • Sakhi A.K.
      • Russnes K.M.
      • Smeland S.
      • Blomhoff R.
      • Gundersen T.E.
      Simultaneous quantification of reduced and oxidized glutathione in plasma using a two-dimensional chromatographic system with parallel porous graphitized carbon columns coupled with fluorescence and coulometric electrochemical detection.
      The redox potential ratio between GSH and GSSG was calculated with the Nernst equation as described by Schafer and Buettner
      • Schafer F.Q.
      • Buettner G.R.
      Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple.
      :
      GSSG+2H++2e2GSH


      Ehc=240(59.1/2)*log([GSH]2/[GSSG])mV


      Here, Ehc is the half-cell reduction potential. The standard potential E0 for the 2GSH/GSSG couple was −264 mV, based on the value of −240 mV for pH 7.0, and a pH effect of −59 mV/pH unit.

      Measurement of Plasma Thiols

      The levels of total GSH, cysteine (Cys), homocysteine (Hcy), and cysteinyl-glycine were analyzed using the Homocysteine by HPLC kit.a Calibration, sample preparation, and HPLC were performed as described elsewhere.
      • Bohn S.K.
      • Smeland S.
      • Sakhi A.K.
      • et al.
      Post-radiotherapy plasma total glutathione is associated to outcome in patients with head and neck squamous cell carcinoma.

      Measurement of Urinary F2-Isoprostane and Creatinine

      The determination of the isoprostane 8-epi-PGF2α was performed by liquid chromatography–mass spectrometry, as described by Bastani et al.
      • Bastani N.E.
      • Gundersen T.E.
      • Blomhoff R.
      Determination of 8-epi PGF(2alpha) concentrations as a biomarker of oxidative stress using triple-stage liquid chromatography/tandem mass spectrometry.
      The creatinine level in urine used for the normalization of 8-epi-PGF2α concentration (8-epi-PGF2α/mg creatinine) was measured at the Department of Medical Biochemistry, Oslo University Hospital, Ullevål.

      Measurement of Plasma Carotenoids

      For analysis of carotenoids (lutein, zeaxanthin, β-kryptoxanthin, α-carotene, β-carotene, and lycopene) in plasma, HPLC with ultraviolet detection was used. Proteins in plasma samples were precipitated by the addition of a 4.5 times volume of isopropanol containing 5µg/mL astaxanthin (internal standard). Samples were mixed for 10 minutes and then centrifuged (3000×g at 4°C) for 15 minutes. Aliquots of 20μL of the supernatant were injected into the HPLC system. The mobile phases consisted of A (20% water and 24% acetone in ethanol) and B (100% acetone). The separation was achieved with a linear gradient from 2% to 100% mobile phase B within 20 minutes, followed by 100% mobile phase B for 15 minutes. The flow rate was 0.3mL/min. Detection was performed at 453nm. Plasma calibrators and controls were quantified against the standardized reference material 968c from the National Institute of Standards and Technology.

      Measurement of Plasma Vitamin E

      For the determination of tocopherols by HPLC, proteins were precipitated by the addition of 3 volumes of isopropanol containing .75μg/mL tocol (internal standard), followed by centrifugation at 3000×g at 4°C for 15 minutes. Then, 5μL of the clear supernatant was used for analysis by HPLC. Separation was achieved using Metachem Taxsil column (4.6×250mm), and the mobile phase consisted of 10% water in methanol. Standards prepared in 1% bovine serum albumin in phosphate buffered saline were used for quantification.

      Statistical Analyses

      The statistical analyses were performed with SPSS version 13–18.0b and MedCalc Software.c The P values less than .05 were considered statistically significant. Data are reported as mean (95% confidence intervals) unless otherwise stated. To assess differences between patients and controls at each time point, linear regression was used, adjusting for smoking, sex, body mass index, and age, because of the possible effects from these parameters on antioxidant levels.
      • Sakhi A.K.
      • Russnes K.M.
      • Thoresen M.
      • et al.
      Pre-radiotherapy plasma carotenoids and markers of oxidative stress are associated with survival in head and neck squamous cell carcinoma patients: a prospective study.
      • Van Herpen-Broekmans W.M.
      • Klopping-Ketelaars I.A.
      • Bots M.L.
      • et al.
      Serum carotenoids and vitamins in relation to markers of endothelial function and inflammation.
      • Preston A.M.
      • Rodriguez C.
      • Rivera C.E.
      Plasma ascorbate in a population of children: influence of age, gender, vitamin C intake, BMI and smoke exposure.
      • Galan P.
      • Viteri F.E.
      • Bertrais S.
      • et al.
      Serum concentrations of beta-carotene, vitamins C and E, zinc and selenium are influenced by sex, age, diet, smoking status, alcohol consumption and corpulence in a general French adult population.
      Changes over time and changes between groups over time were evaluated with a linear mixed model.

      Results

      The characteristics of the study population and controls and the inclusion process of SCI patients are shown in table 1 and figure 1, respectively.
      Table 1Characteristics of the Study Populations at Baseline
      VariablesLevel of SCI Tetraplegic (n= 7)Level of SCI Paraplegic (n= 9)Level of SCI Incomplete (n= 21)All SCI (n= 37)Controls (n= 346)
      Age (y)33 (18–50)37 (18–61)42 (19–60)39
      P<.05 between the SCI and control groups.
       (18–61)
      46 (18–80)
      BMI (kg/m2)26 (23.0–29.0)26 (21.3–32.5)26.8 (21.5–35.1)26.4 (21.3–35.5)25 (15.7–39.6)
      Male671831169
      Female1236
      P<.05 between the SCI and control groups.
      178
      Smokers117959
      NOTE. Values are means (95% confidence intervals).
      Abbreviation: BMI, body mass index.
      low asterisk P<.05 between the SCI and control groups.
      Figure thumbnail gr1
      Fig 1Flowchart of the inclusion process of the SCI subjects.
      In an initial analysis using linear regression, we compared the biomarkers in subjects with complete tetraplegia, complete paraplegia, or incomplete tetraplegia or paraplegia at all the 3 time points as well as possible changes of the biomarkers within each subgroup using a linear mixed model. We found no significant differences in biomarkers between the 3 subgroups at any of the time points studied during the first year after injury, except for probable random observations of significance at 1 time point of lutein, α-carotene, total GSH, and cysteinyl-glycine (Cys/Gly) (see Supplemental Table 1, Supplemental Table 2, Supplemental Table 3, available online only at www.archives-pmr.org). Furthermore, only lutein (P=.04) showed a different trend of change within complete tetraplegic patients. Therefore, data from all of the 37 SCI patients were pooled for each of the 3 time points in all further analyses.

      Antioxidants and Oxidative Stress Biomarkers 1 Month After Injury

      The mean levels of plasma and urine biomarkers of antioxidant status and oxidative stress obtained from the SCI patients and the able-bodied controls are presented in table 2. At the earliest time point (eg, 1mo after injury), plasma total GSH and total Cys/Gly were reduced by 28% and 19%, respectively, compared with the controls. The oxidation product GSSG and the redox potential were also reduced (ie, 42% and 7%, respectively).
      Table 2Plasma and Urine Concentrations of Endogenous Biomarkers
      BiomarkerTime Point 1 (n=37)Time Point 2 (n=26)Time Point 3 (n=33)Controls (n=346)P
      P values for trend between time points in the SCI patients.
      Redox potential (mV)–139.5 (−163 to −117)–138.0 (−153 to −125)–138.6 (−173 to −122)–150.5 (−173 to −128).740
      P<.001
      P values for SCI versus controls.
      P<.001
      P values for SCI versus controls.
      P<.001
      P values for SCI versus controls.
      Total GSH (μmol/L)3.7 (1.3 to 13.5)3.19 (1.3 to 5.6)4.54 (1.6 to 9.4)5.2 (2.6 to 10.2).001
      P<.001P<.001P<.13
      Total Cys (μmol/L)244.7 (191.0 to 428.0)234.0 (180.0 to 320.0)221.6 (147.0 to 305.0)242.6 (165.8 to 326.5).180
      P<.99P<.19P<.011
      Total Cys/Gly (μmol/L)18.2 (11.5 to 33.0)17.2 (12.3 to 27.3)17.4 (9.8 to 26.0)22.4 (14.0 to 31.0).730
      P<.001P<.001P<.001
      Total Hcy (μmol/L)9.3 (4.3 to 17.2)11.0 (5.2 to 26.0)11.3 (4.5 to 21.0)9.7 (4.7 to 17.5).001
      P<.48P<.22P<.11
      GSSG (μmol/L)23.3 (3.0 to 87.0)21.0 (8.4 to 54.0)33.5 (3.6 to 63.0)40.0 (6.4 to 99.5).001
      P<.001P<.001P<.16
      GSH (μmol/L)1.3 (0.6 to 3.5)1.1 (0.5 to 2.9)1.5 (0.8 to 2.9)2.4 (1.0 to 4.4).001
      P<.001P<.001P<.001
      8-ePi PGF2α (pg/mg cr)388.8 (27.0 to 1340.0)328.6 (51.0 to 920.0)498.6 (39.0 to 3362.0)241.8 (48.3 to 690.0).330
      P<.001P<.001P<.001
      NOTE. Values are means (95% confidence intervals).
      Abbreviation: cr, creatinine.
      low asterisk P values for SCI versus controls.
      P values for trend between time points in the SCI patients.
      Furthermore, the SCI patients had 38% to 71% lower levels of all the 6 carotenoids (lutein, zeaxanthin, β-kryptoxanthin, α-carotene, β-carotene, and lycopene) and vitamin E (α-tocopherol) compared with the controls 1 month after injury.
      The oxidative stress biomarker urinary 8-epi PGF2α had increased by 161% in the SCI patients compared with the controls.

      Antioxidants and Oxidative Stress Biomarkers After 3 and 12 Months Follow-Up

      After 3 and 12 months follow-up, most of the biomarkers were still significantly reduced compared with the controls (see Table 2, Table 3). By using a linear mixed model, we analyzed changes over time during the 12 months of follow-up. We observed that total and free GSH improved, while no trend was observed with regard to the redox potential, total Cys, or total Cys/Gly. Interestingly, plasma total Hcy, which has been linked to a number of cardio- and cerebrovascular diseases,
      • Beccia M.
      • Mele M.C.
      • Ferrari M.
      • Ranieri M.
      • Barini A.
      • Rasura M.
      Young stroke and basal plasma and post-methionine load homocysteine and cysteine levels 1 year after the acute event: do plasma folates make the difference.
      increased significantly during the first year of follow-up.
      Table 3Plasma and Urine Concentrations of Exogenous or Dietary Biomarkers
      BiomarkerTime Point 1 (n=37)Time Point 2 (n=26)Time Point 3 (n=33)Controls (n=346)P
      P values for trend between time points in the SCI patients.
      Carotenoids (nmol/L)
       Lutein72.4 (22.0–170.0)
      P values for SCI versus controls.
      82.7 (20.0–170.0)
      P values for SCI versus controls.
      114.0 (40.0–364.0)
      P values for SCI versus controls.
      167.6 (69.3–346.0).001
      P<.001P<.001P<.001
       Zeaxanthin26.3 (10.0–61.0)28.0 (13.0–50.0)27.9 (11.3–53.0)43.0 (14.7–91.0).64
      P<.001P<.004P<.001
       β-kryptoxanthin78.0 (13.5–227.0)110.4 (18.0–440.0)91.4 (26.0–366.0)164.0 (27.7–476.4).620
      P<.001P<.11P<.016
       α-carotene40.0 (6.8–100.5)49.0 (7.3–12.0)58.3 (9.0–249.0)137.3 (19.0–493.5).020
      P<.001P<.001P<.001
       β-carotene203.4 (45.0–85.0)236.5 (103.0–590.0)255.5 (55.0657.0)507.5 (114.0–1398.0).130
      P<.001P<.001P<.004
       Lycopene336.0 (100.0–753.0)461.8 (74.0–770.0)431.0 (150.0–985.0)633 (134.0–1323.0).150
      P<.001P<.002P<.001
      Vitamin E (μmol/L)
       α-tocopherol22.5 (13.4–35.4)22.7 (16.4–30.6)21.3 (13.3–31.7)30.0 (16.0–51.6).140
      P<.001P<.001P<.001
       δ-tocopherol1.6 (0.6–4.4)1.8 (0.8–3.3)1.8 (0.5–6.2)NA.120
      NOTE. Values are means (95% confidence intervals).
      Abbreviation: NA, not assessed.
      low asterisk P values for SCI versus controls.
      P values for trend between time points in the SCI patients.
      The mean plasma levels of all the carotenoids (lutein, zeaxanthin, α-carotene, β-carotene, and lycopene) and vitamin E (α-tocopherol) were 35% to 64% lower than the controls, while the oxidative stress biomarker, urinary 8-epi PGF2α, had increased to 208% in the SCI patients compared with the controls after 12 months.
      Additionally, a trend analysis demonstrated that lutein and α-carotene increased during the first year after injury, while zeaxanthin, β-carotene, lycopene, and α-tocopherol did not improve among the SCI patients.

      Discussion

      This report is to our knowledge the first controlled study that examines the plasma levels of endogenous and exogenous antioxidant defense biomarkers as well as an oxidative stress biomarker in SCI patients during the early phase (ie, 1–12mo) after injury. Several plasma antioxidant biomarkers were decreased while the urine-derived biomarker of oxidative stress, 8-epi PGF2α, was increased 1 month after injury. Some of the biomarkers improved during the first year after injury, but biomarkers such as the redox potential, plasma α-tocopherol, and most of the plasma carotenoids did not. The oxidative stress biomarker as well as Hcy, a biomarker for cardio- and cerebrovascular diseases, increased significantly during the first year of follow-up.
      GSH is a major endogenous cellular antioxidant. Plasma GSH and its conjugated and oxidized forms are biomarkers of cellular GSH status. The decreased GSH concentration immediately after the injury (1 and 3mo) in SCI patients indicates that the acute trauma depletes the GSH pool, possibly because of oxidative stress caused by an ongoing inflammation. The plasma levels of both reduced and total GSH increased from 3 months to 1 year after injury. However, the levels were still lower than among the controls, indicating that SCI patients may be vulnerable to oxidative stress, even 1 year after injury. These observations are in accordance with a study using an SCI model in rats, which showed that GSH decreased significantly at an early stage of SCI.
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      • Griebel R.W.
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      Promoting glutathione synthesis after spinal cord trauma decreases secondary damage and promotes retention of function.
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      • et al.
      Diurnal variation in glutathione and cysteine redox states in human plasmas.
      However, during a massive or a prolonged type of oxidative stress where the GSH pool is depleted, reduced plasma concentrations of GSSG are often observed.
      • Bohn S.K.
      • Smeland S.
      • Sakhi A.K.
      • et al.
      Post-radiotherapy plasma total glutathione is associated to outcome in patients with head and neck squamous cell carcinoma.
      Frequently, the ratio of GSH and GSSG is used as a biomarker of oxidative stress.
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      • Yang S.
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      • Turner N.D.
      Glutathione metabolism and its implications for health.
      However, several recent studies have emphasized the importance of the redox state of the GSH/GSSG couple when calculated using the Nernst equation, as described by Schafer and Buettner.
      • Schafer F.Q.
      • Buettner G.R.
      Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple.
      Our finding of a lowered redox potential (ie, redox state of GSH/GSSG) at 1, 3, and 12 months follow-up indicates increased oxidative stress in the SCI patients. This is also in accordance with the increased urine concentrations of 8-epi PGF2α in the SCI subjects in the present, as well as other studies.
      • Oner-Iyidogan Y.
      • Kocak H.
      • Gurdol F.
      • Kocak T.
      • Erol B.
      Urine 8-isoprostane F-2 alpha concentrations in patients with neurogenic bladder due to spinal cord injury.
      • Cracowski J.L.
      • Durand T.
      • Bessard G.
      Isoprostanes as a biomarker of lipid peroxidation in humans: physiology, pharmacology and clinical implications.
      • Tarcan T.
      • Siroky M.B.
      • Krane R.J.
      • Azadzoi K.M.
      Isoprostane 8-epi PGF2 alpha, a product of oxidative stress, is synthesized in the bladder and causes detrusor smooth muscle contraction.
      We observed that exogenous antioxidants (ie, carotenoids and α-tocopherol) in SCI subjects were lower compared with controls. Furthermore, zeaxanthin, α-carotene, lycopene, and α-tocopherol did not improve in SCI patients the first year after injury. This is in accordance with previous observations of low levels of plasma carotenoids and vitamin E in SCI subjects.
      • Burri B.J.
      • Dopler-Nelson M.
      • Neidllinger T.R.
      Measurements of the major isoforms of vitamins A and E and carotenoids in the blood of people with spinal-cord injuries.
      These results suggest that SCI patients have a reduced intake of fruits and vegetables and/or increased use of these antioxidants.

      Study Limitations

      The major strengths of this study are the well-characterized cohort of SCI subjects and the inclusion of repetitive measurements of a large panel of biomarkers for antioxidant status and oxidative stress. However, a potential weakness is the lack of dietary records that potentially could have supported our findings on dietary antioxidants. In addition, a formal sample size calculation was not performed in the present study because of a lack of background data and the limited number of eligible SCI subjects.

      Conclusions

      Our data demonstrate lowered levels of dietary and endogenous antioxidants in SCI subjects compared with able-bodied controls, simultaneously with increased oxidative stress. These observations indicate an association between SCI and lowered antioxidant defense, probably caused by increased levels of oxidative stress the first year after an acute injury to the spinal cord. This is further supported by the observation that the level of lipid peroxidation biomarker, 8-epi PGF2α, was increased while the plasma redox potential was reduced in the SCI patients. Collectively, these observations suggest that therapeutic interventions to dampen oxidative stress, for example by dietary supplements of antioxidants, might be beneficial in acute SCI.
      • a
        BioRad Laboratories, Heidemannstr. 164, 80939 Munich, Germany.
      • b
        SPSS Inc, 233 S Wacker Dr, 11th Fl, Chicago, IL 60606.
      • c
        MedCalc Software, Broekstraat 52, 9030 Mariakerke, Belgium.

      Appendix

      Supplemental Table 1Change of Biomarkers Within Groups at Time Point 1, 1 Month After Injury
      BiomarkerGroup AGroup BGroup CP
      P values for group A versus group B.
      P
      P values for group A versus group C.
      P
      P values for group B versus group C.
      Carotenoids (nmol/L)
       Lutein73.5 (36.0 to 170.0)62.3 (42.0 to 100.0)76.3 (22.0 to 150.0).30.560.47
       Zeaxanthin23.4 (12.7 to 60.0)26.0 (13.0 to 40.0)27.4 (10.0 to 60.0).08.240.83
       β-kryptoxanthin63.6 (20.0 to 130.0)92.7 (24.5 to 230.0)76.5 (13.5 to 200.0).50.800.11
       α-carotene38.7 (25.5 to 60.0)31.6 (11.5 to 40.0)42.9 (6.8 to 100.0).44.650.32
       β-carotene209.0 (93.7 to 410.0)265.3 (44.9 to 920.0)174.9 (74.0 to 370.0).67.270.33
       Lycopene389.0 (170.0 to 790.0)314.0 (210.0 to 430.0)327.2 (0 to 520.0).75.740.82
      Vitamin E (μmol/L)
       α-tocopherol22.0 (15.5 to 28.1)22.1 (15.9 to 27.9)22.8 (13.3 to 35.9).70.780.87
       δ-tocopherol1.7 (0.6 to 3.0)2.0 (0.6 to 4.6)1.4 (0.6 to 2.8).53.600.41
      Redox potential (mV)−138.8 (−146 to –134)−144.4 (−163 to –129)−137.5 (−163 to –117).58.760.92
      Total GSH (μmol/L)2.5 (1.8 to 3.5)4.5 (1.3 to 13.6)3.8 (1.7 to 9.4).40.095.96
      Total Cys (μmol/L)248.0 (192.8 to 307.5)236.0 (193.4 to 264.0)248.1 (191.2 to 428.2).25.910.99
      Total Cys/Gly (μmol/L)16.6 (13.5 to 19.1)18.0 (13.7 to 26.5)19.0 (11.5 to 33.4).50.200.56
      Total Hcy (μmol/L)9.3 (4.3 to 15.7)9.3 (4.3 to 13.6)9.3 (5.4 to 17.2).78.900.47
      GSSG (μmol/L)13.4 (11.0 to 17.2)21.7 (3.3 to 35.2)27.0 (3.0 to 91.0).15.320.22
      GSH (μmol/L)0.9 (0.7 to 1.1)1.5 (0.7 to 3.6)1.2 (0.6 to 2.4).36.070.46
      8-epi PGF2α (pg/mg cr)448.4 (94.0 to 1357.0)330.7 (27.0 to 664.0)385.5 (41.0 to 1154.0).64.220.66
      NOTE. Values are means (95% confidence intervals).
      Abbreviations: cr, creatinine; Group A, complete tetraplegia (AIS grade A); Group B, complete paraplegia (AIS grade A); Group C, incomplete tetraplegia or paraplegia (AIS grade D).
      low asterisk P values for group A versus group B.
      P values for group A versus group C.
      P values for group B versus group C.
      Supplemental Table 2Change of Biomarkers Within Groups at Time Point 2, 3 Months After Injury
      BiomarkerGroup AGroup BGroup CP
      P values for group A versus group B.
      P
      P values for group A versus group C.
      P
      P values for group B versus group C.
      Carotenoids (nmol/L)
       Lutein84.4 (56.9 to 130.0)80.4 (42.2 to 130.0)136.0 (52.1 to 272.0).55.63.920
       Zeaxanthin30.0 (17.0–50.0)31.0 (18.0 to 50.0)29.8 (11.3 to 50.0).61.38.870
       β-kryptoxanthin143.3 (18.0 to 440.0)111.5 (66.0 to 190.0)86.4 (26.0 to 280.0).52.62.360
       α-carotene52.3 (26.6 to 70.0)43.8 (14.1 to 90.0)61.3 (9.0 to 200.0).33.53.240
       β-carotene209.4 (123.5 to 260.0)267.0 (110.9 to 590.0)244.1 (54.9 to 600.0).54.77.940
       Lycopene491.5 (327.9 to 760.0)464.8 (283.3 to 770.0)458.2 (166.5 to 1000.0).92.76.930
      Vitamin E (μmol/L)
       α-tocopherol22.3 (18.0 to 28.7)22.3 (16.4 to 30.6)22.3 (14.5 to 31.7).74.92.700
       δ-tocopherol1.7 (0.8 to 3.3)2.0 (1.1 to 3.0)1.6 (0.6 to 2.7).33.41.740
      Redox potential (mV)−134.7 (−149 to –125)−138.1 (−153 to –125)−139.0 (−173 to –129).94.55.340
      Total GSH (μmol/L)2.4 (1.3 to 3.9)3.6 (1.3 to 5.6)4.9 (2.3 to 9.4).25.59.021
      Total Cys (μmol/L)259.0 (189.0 to 320.4)211.0 (180.3 to 242.8)226.0 (183.2 to 306.1).13.65.400
      Total Cys/Gly (μmol/L)16.3 (12.7 to 19.1)15.2 (12.3 to 18.2)19.1 (13.0 to 26.0).47.12.450
      Total Hcy (μmol/L)14.0 (6.3 to 26.1)9.0 (5.6 to 11.2)11.3 (4.5 to 21.0).20.48.450
      GSSG (μmol/L)15.0 (8.4 to 25.3)23.0 (10.5 to 54.0)34.6 (3.6 to 62.0).20.39.430
      GSH (μmol/L)0.8 (0.6 to 1.1)1.3 (0.6 to 3.0)1.5 (0.8 to 2.3).40.80.120
      8-epi PGF2α (pg/mg cr)385.0 (146.0 to 920.0)355.0 (94.0 to 688.0)593.6 (39.0 to 3362.0).26.45.490
      NOTE. Values are means (95% confidence intervals).
      Abbreviations: cr, creatinine; Group A, complete tetraplegia (AIS grade A); Group B, complete paraplegia (AIS grade A); Group C, incomplete tetraplegia or paraplegia (AIS grade D).
      low asterisk P values for group A versus group B.
      P values for group A versus group C.
      P values for group B versus group C.
      Supplemental Table 3Change of Biomarkers Within Groups at Time Point 3, 12 Months After Injury
      BiomarkerGroup AGroup BGroup CP
      P values for group A versus group B.
      P
      P values for group A versus group C.
      P
      P values for group B versus group C.
      Carotenoids (nmol/L)
       Lutein87.3 (40.0 to 130.0)90.3 (48.8 to 200.0)136.0 (52.1 to 272.0).15.02.06
       Zeaxanthin25.0 (12.0 to 40.0)26.4 (13.7 to 50.0)29.8 (11.3 to 50.0).33.37.97
       β-kryptoxanthin92.4 (28.4 to 250.0)99.7 (29.4 to 370.0)86.4 (26.0 to 280.0).54.08.06
       α-carotene79.0 (36.1 to 250.0)40.6 (11.0 to 80.0)61.3 (9.0 to 200.0).74.41.03
       β-carotene261.7 (152.5 to 550.0)272.4 (136.5 to 660.0)244.1 (54.9 to 600.0).42.24.13
       Lycopene408.3 (265.6 to 60.0)395.7 (147.7 to 600.0)458.2 (166.5 to 1000.0).84.28.32
      Vitamin E (μmol/L)
       α-tocopherol20.1 (13.4 to 27.4)20.0 (14.4 to 25.6)22.3 (14.5 to 31.7).18.50.60
       δ-tocopherol1.3 (0.7 to 1.3)2.4 (0.5 to 6.3)1.6 (0.6 to 2.7).47.44.30
      Redox potential (mV)−136.0 (−138 to –133)−139.7 (−150 to –122)−139.0 (−173 to –129).70.86.92
      Total GSH (μmol/L)3.1 (1.6 to 4.5)4.7 (2.2 to 7.9)4.9 (2.3 to 9.4).18.60.09
      Total Cys (μmol/L)205.3 (147.1 to 258.0)224.0 (165.4 to 252.8)226.0 (183.2 to 306.1).44.87.24
      Total Cys/Gly (μmol/L)14.5 (9.8 to 21.1)15.8 (12.4 to 21.5)19.1 (13.0 to 26.0).28.05.08
      Total Hcy (μmol/L)10.5 (4.6 to 20.8)11.0 (7.6 to 17.0)11.3 (4.5 to 21.0).90.83.43
      GSSG (μmol/L)21.5 (12.6 to 41.5)38.7 (15.3 to 63.0)34.6 (3.6 to 62.0).40.36.47
      GSH (μmol/L)1.0 (0.8 to 1.3)1.7 (0.8 to 3.0)1.5 (0.8 to 2.3).55.47.27
      8-epi PGF2α (pg/mg cr)262.6 (78.0 to 443.0)576.1 (127.0 to 1169.0)593.6 (39.0 to 3362.0).83.88.70
      NOTE. Values are means (95% confidence intervals).
      Abbreviations: cr, creatinine; Group A, complete tetraplegia (AIS grade A); Group B, complete paraplegia (AIS grade A); Group C, incomplete tetraplegia or paraplegia (AIS grade D).
      low asterisk P values for group A versus group B.
      P values for group A versus group C.
      P values for group B versus group C.

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