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Oxidative Balance in Lymphocytes From Patients With Nonalcoholic Steatohepatitis
CLINICAL INVESTIGATION
Oxidative Balance in Lymphocytes From Patients With Nonalcoholic Steatohepatitis Silvia Belia, PhD, Graziana Lupattelli, MD, Eleonora Urbani, PhD, Gaetano Vaudo, MD, Anna Rita Roscini, MD, Stefano Perni, PhD and Valeria Marsili, PhD
Abstract: Oxidative stress is linked to several human diseases, including nonalcoholic steatohepatitis (NASH). In this study, lymphocytes were used as a model to study this disease. These cells offer several advantages for cellular and molecular studies such as easy accessibility, and they are easily accessible and constitute a “time-persistent” system capable of reflecting the condition of the whole organism. Lymphocytes from patients with NASH display oxidative stress features. Among the possible causes for the overproduction of reactive oxygen species in NASH lymphocytes, there might be alterations of enzymatic pathways, autooxidation of glucose and mitochondrial superoxide production, which, in turn, would lead to protein oxidative damage. Increased oxidative stress in lymphocytes from patients with NASH may result in a pro-oxidative environment, which, in turn, could modify the pathway of the enzymatic activities. The data confirm that an imbalance between pro-oxidant and antioxidant defense mechanisms may be an important factor in NASH. Key Indexing Terms: Oxidative stress; Lymphocytes; Metabolic syndrome; NAFLD; ROS. [Am J Med Sci 2014;348(1):30–36.]
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onalcoholic fatty liver disease (NAFLD) is defined as fat accumulation in the liver exceeding 5% to 10% by weight, as microscopically determined from the percentage of fat-laden hepatocytes. The diagnosis of NAFLD implies the exclusion of the other causes of steatosis, such as viruses, alcohol, autoimmunity, toxicity, hypobetalipoproteinemia and Wilson’s disease. Nonalcoholic steatohepatitis (NASH) is the advanced form of NAFLD that progresses from simple steatosis to liver inflammation,1,2 fibrosis, cirrhosis and, possibly, hepatocarcinoma.3 NAFLD/NASH are strictly linked with obesity and insulin resistance and are frequently associated with hypertension and dyslipidemias, a constellation of abnormalities known as metabolic syndrome, and in many cases with type 2 diabetes mellitus. Metabolic syndrome is also a risk factor for NASH and for advanced fibrosis in liver tissue.4 Taken together, NAFLD and NASH affect between 9% and 33% of the general population in the developed countries. However, although NAFLD is considered a common and relatively benign liver condition, NASH is thought to potentially lead to morbidity and increased mortality, thus demanding more attention. The pathogenesis of NASH is thought to be multifactorial: the inducing factors include oxidative stress, iron deposits, overexpression of cytochrome P450E1, participation of endotoxin and tumor necrosis factor-a and mitochondrial disorders.
From the Departments of Cellular and Environmental Biology (SD, EU, SP, and Clinical and Experimental Medicine, Internal Medicine, Angiology and Atherosclerosis (GL, GV, ARR), University of Perugia, Perugia, Italy. Submitted April 24, 2013; accepted in revised form August 7, 2013. The authors have no financial or other conflicts of interest to disclose. Correspondence: Silvia Belia, PhD, Department of Cellular and Environmental Biology, University of Perugia, Via Elce di Sotto, Perugia 06123, Italy (E-mail: fi[email protected]).
Some authors have proposed the so-called “two-hit” model to describe the underlying mechanism. The “first hit” involves the accumulation of triglycerides within hepatocytes and the “second hit” regard oxidative stress that leads to inflammation, cellular injury and progressive fibrosis.5 Abnormal mitochondrial and cellular redox homeostasis has been documented in cases of steatohepatitis, which suggests that there are alterations in the signaling cascades, which would ultimately alter the function of the enzymes and proteins that are critical for mitochondrial and cellular function.6 Mitochondria play a major role in fat oxidation and energy production. They are also the main source of the reactive oxygen species (ROS) that trigger lipid peroxidation, cytokine overproduction and cell death.7 The products of ROS-induced lipid peroxidation impair the respiratory chain, leading to increased mitochondrial ROS formation, which could deplete the antioxidants and impair ROS inactivation.8 In patients with NASH, insulin resistance is the pathogenic factor that favors the accumulation of free fatty acids in the liver. Insulin resistance is also believed to induce oxidative stress by stimulating microsomal lipid peroxidases and to decrease mitochondrial beta-oxidation because of the direct effect of high insulin levels.3 To evaluate whether the pathogenesis of NASH is associated with increased levels of ROS, the following had to be ascertained: (1) significant accumulation of ROS derived from used cell metabolism; (2) definite oxygen radical damage; and (3) abnormalities in the antioxidant defenses in pathological patients. The validation of these conditions was necessary to establish the possible relationship with cascade signaling events directly correlated with the pathogenesis of the disease. Lymphocytes from peripheral blood were chosen for this study, because they can act as biosensors of the whole organism in response to the disease. These cells are not only involved in the inflammatory processes and immune responses but also contribute to homeostasis and adaptation to environmental variations and pathological conditions.9 Lymphocytes also have a remarkably long lifetime and have the capacity to keep memory of their original phenotype even after they are cultured. Most importantly, there is no evidence that highly reactive chemical species capable of interacting with biological macromolecules and altering cellular functions have any effect on lymphocytes of patients with NASH.10 For all of these reasons, lymphocytes are a robust, “time-resistant” system capable of monitoring the conditions of the entire body and keeping track of cumulative long-term stressors.9 To evaluate the involvement of oxidative stress in the pathogenesis of NASH, experiments were carried out to aim at verifying the occurrence and the level of the oxidative stress in lymphocytes derived from patients with NASH.
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MATERIALS AND METHODS Materials All media, sera, antibiotics and culture solutions were purchased from Sigma—Aldrich, Corp. (St. Louis, MO). All
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sterile culture plastics were provided by Falcon (Plymouth, United Kingdom). All other reagents were analytical grade. Patients A total of 15 overweight/obese patients (4 women and 9 men; mean age, 42 6 14 years, body mass index [BMI] 30 6 5.4 kg/m2), with a clinical diagnosis of NAFLD were included in the study. The diagnosis of NAFLD was based on the following criteria: (1) elevated aminotransferases (aspartate aminotransferase [AST] and/or alanine aminotransferase) in at least 2 determinations; (2) clinical and hematochemical exclusion of alcohol-induced or drug-induced liver disease, autoimmune or viral hepatitis and cholestatic or metabolic/genetic liver disease; and (3) sonography documenting liver steatosis. A total of 12 age-matched normal weight subjects (5 women and 8 men) from the Hospital staff were included as control group. A complete medical history and clinical examination was performed in all patients and controls. BMI was calculated using the formula: weight (in kilograms)/height (in square meters). Waist circumference was assessed by wrapping the tape around the waist midway between 2 measure points: the top of hip bone and the bottom of the ribs. Blood was drawn in the morning after a 13-hour fast and the following parameters were determined: total cholesterol, triglycerides, high-density lipoprotein-cholesterol, low-density lipoprotein-cholesterol (Friedewald’s formula), glycemia, insulinemia, AST, alkaline phosphatase, g-glutamyl transferase (GT) and apolipoprotein B. Liver Sonography Sonography of the liver was performed by a skilled ultrasonographer, unaware of clinical and laboratory patients details, using real-time l’ATL (Philips Electronics, Koninklijke, The Netherlands) equipped with a 3.5- to 5-MHz probe. Steatosis was defined sonographically by the appearance of hyperechoic liver tissue with fine, tightly packed echoes and posterior beam attenuation. Bioimpedentiometry All patients and controls underwent bioimpedentiometry (50 kHz, amplitude 50 mA; Tanita Europe B.V., Amsterdam, The Netherlands), using electrodes applied to the foot-plantar surface. This methodology allows the measurement of fat mass in kilogram and as percentage of body weight, free fat mass and water in kilogram. Vascular Ultrasound Measurement Carotid and femoral arteries were examined by highresolution B-mode ultrasonography.1 The examination was performed with a commercially available ultrasound device (ESAOTE Technos MP; ESAOTE S.p.A, Genoa, Italy) equipped with a linear multifrequency 7.5- to 12-MHz transducer. Subjects were examined in the supine position, and all measurements were obtained at enddiastole with electrocardiographic triggering. The ultrasound images were stored on a digital support and analyzed using an image processing workstation (AMS System, Gothenburg, Sweden). On a longitudinal 2-dimensional ultrasound scanning, the image of the far wall of common carotid and common femoral artery, at the prebifurcation tract, was displayed as 2 bright white lines separated by a hypoechogenic space. The intima-media thickness (IMT) of the far wall was automatically measured by the computerized system mentioned earlier (IMT common carotid and IMT common femoral). The intraobserver coefficient of variation was 1.1% (mean 6 standard deviation of the difference 0.018 6 0.031 mm), whereas interobserver values were 1.8% (0.028 6 0.032 mm). Ó 2013 Lippincott Williams & Wilkins
Flow-Mediated Vasodilation Flow-mediated vasodilation (FMV) was assessed on the brachial artery by ultrasonography. Any drug known to affect endothelial function, including nitrates, hypolipidemic drugs and aspirin, was withdrawn $1 week before the examination. Details of the procedure, which was performed according to the International Brachial Artery Task Force guidelines,3 have been reported elsewhere.4 Briefly, the measurements were performed in supine position on the nondominant arm, after 10 to 20 minutes resting in a quiet, dark room with a temperature of 22°C. The brachial artery was scanned longitudinally just above the antecubital crease using a 10-MHz probe (HDI 3500; Advanced Technology Laboratories). Diameter of the brachial artery was measured at the R-wave of the electrocardiogram, on the interface between media and adventitia of the anterior and posterior wall. Gain settings were optimized to identify the lumen and the vessel wall interfaces and were not modified during the examination. Hyperemia was induced by inflation of a pneumatic cuff (12.5 cm wide) at 230 to 250 mm Hg for 4 minutes on the most proximal portion of the upper arm. Arterial diameter measurement was repeated 45 to 60 seconds after sudden deflation of the cuff. Tracings were recorded on videotape and read by 1 investigator, who was unaware of the subject’s clinical data and temporal sequence. The average of 3 measurements of basal and posthyperemia diameter was used for the analysis. FMV was expressed as the relative increase in brachial artery diameter during hyperemia, and defined as 100 3 ([posthyperaemia diameter 2 basal diameter]/basal diameter). Blood flow was measured as arterial cross-sectional area (p 3 r2) times mean Doppler velocity corrected for angle. The intraobserver between-occasion reproducibility of FMV in our laboratory was assessed in 10 subjects examined 2 days apart. The mean 6 SD difference between the 2 examinations was 1.0% 6 1.5%.11–14 Isolation of Human Lymphocytes Peripheral venous blood samples from healthy donors and patients with NASH were collected in sodium-heparinized vacutainers. Peripheral blood lymphocytes were separated under sterile conditions on a Ficoll-Paque PLUS (Amersham, Piscataway, NY) gradient using the method by Boyum.15 Aliquots of heparinized whole blood diluted with an equal volume of Dulbecco’s phosphate-buffered saline (1:1) were gently applied to an equal volume of Ficoll-Paque PLUS in centrifuge tubes. Samples were centrifuged at 400g for 30 minutes. The resultant interface (buffy coat) was carefully aspirated from the gradient and washed twice in Dulbecco’s phosphate-buffered saline by centrifugation at 200g for 10 minutes. The subsequent pellet was resuspended in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2% L-glutamine and 1% penicillin/streptomycin. Monocytes were removed from the mononuclear fraction by adherence to Petri dishes during overnight incubation at 37°C. Purified lymphocytes were finally resuspended in complete RPMI 1640 medium (1–2 3 106 cells/mL). Cell viability was determined by Trypan blue dye exclusion. The purified lymphocytes were used for experimental analyses within 1 day from their isolation. ROS Generation Determination of Hydrogen Peroxide Production Hydrogen peroxide (H2O2) generation in lymphocytes from healthy subjects and patients with NASH was assayed using a colorimetric method involving the oxidation
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of iodide in the presence of ammonium molybdate and photometric analyses of the resulting blue starch-iodine complex performed at 570 nm.16 Briefly, human blood lymphocytes were treated with 38.5 mM HCl, 80 mM potassium iodide, 80 mM ammonium molybdate in H2SO4 and 0.38% starch. Twenty minutes after adding potassium iodide, sample absorbance was measured at 570 nm using a Titertek Multiskan MC plate reader (ICN/Flow Biochemicals, Huntsville, AL). The H2O2 concentration was estimated using a standard curve. Results are expressed as micrograms of H2O2 per 10 5 cells. Determination of O22 Release Intracellular superoxide anion (O22) production in lymphocytes from healthy subjects and patients with NASH was assayed as previously described,17 using a colorimetric method based on the reaction between nitroblue tetrazolium chloride and O22, with the consequent formation of formazan salt. Briefly, human blood lymphocytes were incubated with 1 mg/mL nitroblue tetrazolium at 37°C for 3 hours. Next, cells were centrifuged (at 500g for 10 minutes) and the pellets treated with dimethyl sulphoxide at 37°C for 20 minutes. The absorbance of formazan salt was measured at 550 nm using dimethyl sulfoxide as the blank. Scavenger Enzyme Activities The assays were performed using samples obtained from sonicated lymphocytes suspended in 20 mM Naphosphate buffer, pH 7.0, along with 1 mg/mL pepstatin, 1 mg/mL leupeptin and 100 mM phenylmethylsulphonyl fluoride as protease inhibitors and centrifuged at 100,000g for 1 hour at 4°C. Cytosol protein concentrations were measured according to the method of Lowry et al.18
Catalase activity was determined based on the decrease in absorbance because of H2O2 consumption (e 5 20.04 mM21$cm21) measured at 240 nm, according to the method previously described.19 The final reaction volume of 1 mL contained 100 mM Na-phosphate buffer, pH 7.0, 12 mM H2O2 and 70 mg of sample. Superoxide dismutase (SOD) activity was determined using the modified method of L’Abbé and Fischer.20 The final assay volume (1 mL) contained 20 mM Na2CO3, pH 10, 10 mM cytochrome c, 1 mM xanthine and xanthine oxidase. Because the xanthine oxidase activity varies, the amount used for the assay was sufficient to stimulate cytochrome c reduction at 550 nm at a rate of 0.025/min without SOD addition. SOD units were calculated on the basis of the definition that 1 unit represents the activity that inhibits cytochrome c reduction by 50%. Glutathione reductase (GR) activity was measured based on the decrease in absorbance induced by NADPH oxidation at 340 nm (e 5 26.22 mM21$cm21).21 The assay mixture contained 100 mM Na-phosphate buffer, pH 7.0, 1 mM glutathione disulphide, 60 mM NADPH and 100 mg of sample in a final volume of 1 mL. Glutathione peroxidase activity was calculated using the method of Lawrence and Burk,22 which involves the measurement of glutathione disulphide formation using a coupled enzyme system with GR. NADPH oxidation was recorded at 340 nm (e 5 26.22 mM21$cm21). Selenium dependence was determined using H2O2 as the substrate. The final reaction volume of 1 mL contained 100 mM Na-phosphate buffer, pH 7.5, 1 mM EDTA, 1 mM NaN3, 2 mM GSH, 1 U GR, 0.24 mM NADPH, 30 to 80 mg sample and 0.6 mM H2O2. Oxidative Damage of Protein Substrates The protein carbonyl content was assayed by reacting 2,4-dinitrophenylhydrazine (DNPH) and protein carbonyls.
TABLE 1. Clinical and laboratory characteristics of patients with NASH and healthy controls Parameters Controls,12 (mean 6 SD) Patients with NASH,15 (mean 6 SD) Age (yr) Waist circumference (cm), M Waist circumference (cm), F BMI (kg/m2) Fat (%) SBP (mm Hg) DBP (mm Hg) Glycemia (mg/dL) Insulin (mU/L) Total cholesterol (mg/dL) LDL-cholesterol (mg/dL) HDL-cholesterol (mg/dL) Triglycerides (mg/dL) Apoprotein B (mg/dL) Serum AST (U/L) Serum alanine aminotransferase (U/L) Serum gamma-glutamyl transferase (U/L) IMTCCR (mm) IMTCCL (mm) IMTFCR (mm) IMTFCL (mm) FMV (%)
40 89.1 87 24.07 36.8 127.08 74.92 84.54 8.74 186.92 121 47.3 71.2 63.4 19 20 28 0.83 0.83 0.75 0.76 10.76
6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6
19 7 5 2.2 3.8 16.9 8.6 9 2.2 46.9 40.4 12.8 31.5 11 7.7 8.7 11.9 0.27 0.28 0.2 0.18 1.02
42 96.4 76 30.1 39.71 126.93 74.6 96.27 13.08 217.2 141.9 41.2 208 100.3 40 66 77 1.25 0.64 0.62 0.61 7.9
6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6
14 36 44 5.4 6.5 13.7 8.4 11.1 4.4 57.9 49 11.9 110.2 29.8 15.9 53.9 43.5 1.96 0.18 0.2 0.21 1.4
P 0.06 0.02 0.02 0.01 0.18 0.61 0.83 0.55 ,0.001 0.37 0.45 0.79 ,0.001 0.005 0.01 ,0.001 0.01 0.10 0.43 0.39 0.22 0.03
FMV, flow-mediated vasodilation; HDL, high-density lipoprotein; LDL, low-density lipoprotein; SD, standard deviation; SBP, systolic blood pressure; DBP, diastolic blood pressure; IMTCCR, Intima Media Thickness Common Carotid Right; IMTCCL, Intima Media Thickness Common Carotid Left; IMTFCR, Intima Media Thickness Femoral Common Right; IMTFCL, Intima Media Thickness Femoral Common Left.
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DNPH reacts with protein carbonyls, forming a Schiff base to produce the corresponding hydrazone, which can be analyzed spectrophotometrically.23 Lymphocytes from healthy and NASH subjects were rinsed with phosphate-buffered saline to remove red blood cells and sonicated in 10 volumes of buffer containing 50 mM MES, pH 6.7 and 1 mM EDTA. The sonicated cytosolic fraction was obtained by centrifugation at 10,000g for 15 minutes at 4°C. The supernatant protein concentration was set in the range of 1 to 10 mg/mL. Eight hundred microliters DNPH or 800 mL HCl (2.5 M) was added to 200 mL protein as the blank control. Samples and blanks were left at room temperature for 1 hour in the dark and vortexed every 15 minutes. One milliliter 20% trichloroacetic acid was added to the samples. After incubation for 5 minutes on ice, samples and blanks were centrifuged at 10,000g for 10 minutes at 4°C. The resulting pellet was resuspended in 1 mL of 10% trichloroacetic acid and centrifuged at 10,000g for 10 minutes at 4°C. Next, the pellet was resuspended in 1 mL of ethanol/ethyl acetate mixture (1:1) and centrifuged at 10,000g for 10 minutes at 4°C. This step was repeated 3 times. After the final wash, protein pellets were resuspended in 500 mL of guanidine hydrochloride and centrifuged at 10,000g for 10 minutes at 4°C. The carbonyl content was determined based on supernatant absorbance at 370 nm, using a molar adsorption coefficient for DNPH of 22,000 mM21$cm21. Results are expressed as nanomoles of DNPH per milligram of proteins. Oxidative Damage of Lipidic Substrates Malondialdehyde (MDA) forms an adduct with thiobarbituric acid that can be measured by spectrophotometry. For lipid peroxidation analysis, we used the OXI-TEK TBARS Assay Kit (ZeptoMetrix Corporation; Cat. No. 0801192). We added 100 mL of sodium dodecyl sulfate to 100 mL of samples obtained by lymphocytes sonication, 2.5 mL of thiobarbituric acid buffer reagent, and then, samples were incubated at 95°C for 1 hour. The reaction was stopped by cooling samples to room temperature in an ice bath for 10 minutes. After a centrifugation at 3000 rpm for 15 minutes, the supernatant absorbance was read at 532 nm. The amount of MDA was calculated using a standard curve. Results are expressed as nanomoles of MDA per milligram of proteins. Statistical Analysis Statistical significance was calculated using the Student’s t test for unpaired data.
RESULTS Clinical and laboratory characteristics of patients with NASH and controls are summarized in Table 1. As expected, patients with NASH showed a higher BMI (P 5 0.01), waist circumference (P 5 0.02), triglycerides (P , 0.001), insulin (P , 0.001), serum AST (P , 0.01), alanine aminotransferase (P , 0.001), g-glutamyl transferase (P , 0.001) and apolipoprotein B (P 5 0.005). FMV was significantly lower in the group of patients with NASH (P 5 0.003). No significant differences emerged in IMT, both on carotid and femoral sites. ROS Generation In lymphocytes isolated from patients with NASH, we found ROS (H2O2 and O22) markedly higher than in controls. Specifically, H2O2 generation was higher in NASH lymphocytes (0.534 6 0.047 versus 0.269 6 0.025; P , 0.0001) (Figure 1A); also, a small but significant increase in the Ó 2013 Lippincott Williams & Wilkins
FIGURE 1. Reactive oxygen species generation in lymphocytes derived from patients with NASH, compared with healthy subjects. (A) Hydrogen peroxide (H2O2) production was assayed using a colorimetric method based on the oxidation of iodide in the presence of ammonium molybdate. Values are expressed as optical density (O.D.). (B) Intracellular O22 formation was assessed using the nitroblue tetrazolium reduction assay (see Methods).
concentration of O22 was observed in patients with NASH, compared with healthy subjects (0.228 6 0.073 versus 0.093 6 0.005; P 5 0.0128) (Figure 1B). Scavenger Enzyme Activity Specific activities of the main antioxidant enzymes are shown in Figure 2. Endogenous scavengers exhibited different activity in patients with NASH: a significantly lower activity of soluble catalase, a glutathione-independent H2O2 scavenging enzyme, was observed in patients with NASH when compared with the control group (20.18 6 1.267 versus 38.29 6 3.49, P 5 0.0097) (Figure 2A); on the contrary, SOD, which transforms the superoxide anion radical into oxygen and hydrogen peroxide, did not show any statistically significant difference between controls and NASH (22.89 6 1.078 versus 25.74 6 0.283; P 5 0.0628) (Figure 2B). The GR activity was significantly lower in patients with NASH compared with the controls (25.44 6 0.398 versus 56.17 6 2.388; P 5 0.0002) (Figure 2C). The
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FIGURE 2. Antioxidant enzyme activities: (A) catalase activity was determined according to the method described by Fano` et al,19 based on the decrease in absorbance because of H2O2 consumption (e 5 20.04 mM21$cm21) measured at 240 nm. (B) Superoxide dismutase (SOD) activity was determined using a modified method of L’Abbe´ and Fischer.20 SOD units were calculated using the definition of a SOD unit as the activity that inhibits the rate of cytochrome c reduction by 50%. (C) Glutathione reductase activity was measured according to the rate of decrease in absorbance induced by NADPH oxidation at 340 nm.21 (D) Glutathione peroxidase activity was calculated with the method of Lawrence and Burk,22 which involves the measurement of glutathione disulphide formation using a coupled enzyme system with glutathione reductase.
glutathione-dependent H2O2 scavenger, GPX, displays significantly higher activity in NASH than in the controls (7.93 6 0.669 versus 1.38 6 0.479; P 5 0.0013) (Figure 2D). Oxidative Damage of Protein and Lipid Substrates The oxidative damage of protein substrates, assessed from protein carbonyl levels, was significantly higher in NASH than controls (4.76 6 0.188 versus 2.71 6 0.151; P 5 0.0011) (Figure 3A). By contrast, there was no difference in the oxidative damage of lipids, because reflected by MDA levels, between control and patients with NASH (8.91 6 0.5 versus 9.73 6 0.61; P 5 0.15) (Figure 3B).
DISCUSSION The prevalence of obesity worldwide has increased dramatically worldwide over the last 3 decades. A variety of adverse health outcomes, grouped under the term of metabolic syndrome, include obesity. In this context of obesity/metabolic syndrome, the liver seems to be significantly impacted by fat deposition. It has become clear that obesity and the factors of metabolic syndrome, which encompass glucose dysregulation and dyslipidemia, are key risk factors for the development and progression of NAFLD. Multiple studies have been performed that show the relationship between insulin resistance and NAFLD and have shown that disordered insulin regulation results in multiple, altered metabolic pathways, which ultimately lead to fat deposition and inflammation in the liver.24,25 In addition, patients with metabolic syndrome are
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at high risk for developing cardiovascular disease including myocardial infarction and stroke. Furthermore, a growing body of evidence suggests that the presence of NAFLD is a risk factor for cardiovascular disease independent of the presence of the other features of metabolic syndrome factors.26,27 In this context, the production of free radicals, which form as a result of oxidative stress, is of critical importance. Oxidative stress is an imbalance between ROS production and the ability of cells, or tissues, to detoxify the reactive intermediates or to repair the resulting damage. Oxygen radicals and independent reactive radical oxygen species, and carbon, nitrogen and sulphur radicals have been linked to several human disease conditions, such as atherosclerosis, hypertension, diabetes and cancer.28 Some of the possible causes for ROS overproduction in NASH lymphocytes could be alterations of enzymatic pathways, auto-oxidation of glucose, mitochondrial superoxide production, etc.29 This state, however, produces ROS accumulation, which, in turn, leads to notable protein oxidative damage. The results of this study show increased intracellular concentrations of both O22 and H2O2 in patients with NASH: the increased concentration of superoxide anion is very dependent on mitochondrial metabolism and the structures involved in this syndrome are considered to be of the mitochondrial type.30 No significant SOD activity increase was observed in patients with NASH, and the O22 concentration was very high. The H2O2 concentration increase could, on the one hand, correspond to a reduced catalase-specific activity, or, on the Volume 348, Number 1, July 2014
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FIGURE 3. Oxidative stress markers: (A) protein oxidative damage was measured as the protein carbonyl content. Values are derived from the reaction between DNPH and protein carbonyl. (B) Oxidative damage derived from lipid peroxidation is expressed as relative values (nmol/mg protein) of malondialdehyde.
other hand, it could be because of a combined action of GSH enzymes. In fact, even if GPX activity increases, the concomitant decreased reductase depressed activity does not supply enough substratum to the mechanism, which prevents H2O2 detoxification. As Baskol et al31 reported, in serum from patients with NASH, oxidative stress produces ROS, which induces uncontrolled lipid peroxidation, and xanthine oxidase activity is negatively correlated with SOD and GSPX activity.32 In this study, massive damage was observed in the enzymatic protein molecules involved. This damage can modify their activity resulting in structural damage. This hypothesis is confirmed by increased oxidative damage (formation of protein carbonyls) to the thiol redox state of the proteins investigated.33 Increased oxidative stress in patients with NASH may result in a pro-oxidation environment, which, in turn, could result in decreased antioxidant enzyme activities.34 The data from this study confirm that the imbalance between pro-oxidant and anti-oxidant defense mechanisms may be an important factor in the pathogenesis of NASH. The effect of ROS on the immune system was analyzed, because it responds to pathogen attacks and fights against infections. The liver is a major immune organ and functions mainly in innate immunity.35,36 Kupffer cells (KCs) in the liver can generate proinflammatory cytokines and ROS. These mediators can act locally or systemically, and the immune responses Ó 2013 Lippincott Williams & Wilkins
can lead to hepatocyte injury.37 KCs can generate hepatic ROS, which are involved in the etiology of NAFLD and in the conversion of simple hepatic steatosis to NASH. The mechanism of action of ROS in NASH formation is still unclear. There are 2 hypotheses regarding the role of the KCs in the development of NAFLD: (1) activated KCs produce ROS, which induce insulin resistance, leading to hepatocyte steatosis38; and (2) activated KCs-derived ROS cause lipid peroxidation. Products of this reaction and KC-derived tumor necrosis factor-a can result in hepatocyte injury. Failure to repair this injury can lead to the development of steatohepatitis. Increased levels of ROS in the leukocytes, activation of nuclear factor-kappa B and changes in lymphocyte surface antigen ratio (CD4+/CD8+) were observed in the NASH model rats. The administration of antioxidants, such as spirulina, reduces these variations and may decrease the inflammatory mechanisms, blocking the crosstalk between oxidative stress and inflammation and inhibiting the progression of NASH.1 Many data show that immunological processes in the liver are involved in NASH. Moreover, the percentages of CD4+ and CD8+ lymphocytes and the ratio CD4+/CD8+ provide an assessment of the immune status. This study demonstrated a real normalization of the immune imbalance after treatment with the antioxidant.39 There are many critical steps in NASH pathogenesis at which therapy can intervene. Patients with NASH must have an extremely moderate and healthy lifestyle with continuous exercise, a controlled diet and intake of antioxidant compounds such as vitamins and ursodesoxycholic acid. Many antioxidant drugs have been shown to have a positive effect in controlling enzymatic levels of the liver enzymes.40 Other studies have shown the beneficial effects of bariatric surgery,41 although controlled trials are not available yet.
CONCLUSIONS The number of patients with NASH will probably increase in the future, because this pathology is considered to be the hepatic manifestation of the metabolic syndrome, and the number of overweight individuals is destined to increase. In the future, it will be essential to understand the mechanism of progression from a nonsteatotic liver to an inflamed organ. Although many hypotheses have been proposed regarding the role of oxidative stress in the pathogenesis of NASH, much remains to be investigated. An overload of lipids mainly in the form of triglycerides is present in the liver and proinflammatory cytokines are directed toward the fatty liver. The inflamed state is aggravated by a process of lipotoxicity, which is the formation of ROS that affects the mitochondrial membranes. Impaired antiinflammatory and antioxidant defense mechanisms may be important factors in the pathogenesis of a chronic NASH. ACKNOWLEDGMENTS The authors are grateful to Professor Roberto Buonaurio and Professor Fabio Franciolini for their critical reading of the manuscript. REFERENCES 1. Adler M, Schaffer F. Fatty liver hepatitis and cirrhosis in obese patients. Am J Med 1979;67:811–16. 2. Ludwig J, Viggiano TR, McGill DB, et al. Nonalcoholic steatohepatitis: Mayo Clinic experiences with a hitherto unnamed disease. Mayo Clinic Proc 1980;55:434–8. 3. Adams LAS, Feldstein AE. Non-invasive diagnosis of nonalcoholic fatty liver and nonalcoholic steatohepatitis. J Dig Dis 2011;12:10–6.
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4. Marchesini G, Bugianesi E, Forlani G, et al. Nonalcoholic fatty liver, steatohepatitis and the metabolic syndrome. Hepatology 2003; 37:917–23. 5. Angulo P. Medical progress in nonalcoholic fatty liver disease. N Engl J Med 2002;346:1221–31. 6. Wei Y, Rector RS, Thyfault PJ, et al. Non alcoholic fatty liver disease and mitochondrial disfunction. World J Gastroenterol 2008;14:193–9. 7. Begriche K, Igoudjil A, Pessayre D, et al. Mitochondrial disfunction in NASH: causes, consequences and possible means to prevent it. Mitochondrion 2006;6:1–28. 8. Day CP, James OF. Steatohepatitis: a tale of two “hits”? Gastroenterology 1998;114:842–5. 9. Belia S, Santilli F, Beccafico S, et al. Oxidative-induced membrane damage in diabetes lymphocytes: effects on intracellular Ca2+ homeostasis. Free Radic Res 2009;43:138–48. 10. Mariggio MA, Falone S, Morabito C, et al. Peripheral blood lymphocytes: a model for monitoring physiological adaptation to high altitude. High Alt Med Biol 2010;11:333–42. 11. Vaudo G, Schillaci G, Evangelista F, et al. Arterial wall thickening at different sites and its association with left ventricular hypertrophy in newly diagnosed essential hypertension. Am J Hypertens 2000;13: 324–31. 12. Liang Q, Wendelhag I, Wikstrand J, et al. A multiscale dynamic programming procedure for boundary detection in ultrasonic artery images. IEEE Trans Med Imaging 2000;19:127–42. 13. Corretti MC, Anderson TJ, Benjamin EJ, et al. Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery: a report of the International Brachial Artery Reactivity Task Force. J Am Coll Cardiol 2002;39:257–65. 14. Vaudo G, Marchesi S, Gerli R, et al. Endothelial dysfunction in young patients with rheumatoid arthritis and low disease activity. Ann Rheum Dis 2004;63:31–5. 15. Boyum A. Isolation of lymphocytes, granulocytes and macrophages. Scand J Immunol 1976;(Suppl 5):9–15. 16. M’Bemba-Meka P, Lemieux N, Chakrabarti SK. Role of oxidative stress, mitochondrial membrane potential, and calcium homeostasis in nickel sulfate-induced human lymphocyte death in vitro. Chem Biol Interact 2005;156:69–80. 17. Rauen U, Petrat F, Li T, et al. Hypothermia injury/cold-induced apoptosis: evidence of an increase in chelatable iron causing oxidative injury in spite of low O2-/H2O2 formation. FASEB J 2000;14:1953–64. 18. Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265–75. 19. Fanò G, Mecocci P, Belia S, et al. Age and sex influence on oxidative damage and functional status in human skeletal muscle. J Muscle Res Cell Motil 2001;22:345–51. 20. L’Abbe MR, Fischer PW. An automated method for the determination of Cu, Zn-superoxide dismutase in plasma and erythrocytes using an ABA-200 discrete analyzer. Clin Biochem 1986;19:175–8. 21. Ramos-Martinez JI, Bartolome TR, Pernas RV. Purification and properties of glutathione reductase from hepatopancreas of mytilus edulis. Comp Biochem Physiol 1983;75B:689–92.
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22. Lawrence RA, Burk RF. Glutathione peroxidase activity in seleniumdeficient rat liver. Biochem Biophys Res Commun 1976;71:952–8. 23. Reznick AZ, Packer L. Oxidative damage to proteins: spectrophotometric method for carbonyl assay. Methods Enzymol 1994;233:357–63. 24. Choudhury J, Sanyal AJ. Insulin resistance and the pathogenesis of nonalcoholic fatty liver disease. Clin Liver Dis 2004;8:575–94. 25. Diehl AM, Clarke J, Brancati F. Insulin resistance syndrome and nonalcoholic fatty liver disease. Endocr Pract 2003;9(Suppl 2):93–6. 26. Hamaguchi M, Kojima T, Takeda N, et al. Nonalcoholic fatty liver disease is a novel predictor of cardiovascular disease. World J Gastroenterol 2007;13:1579–84. 27. Choi SY, Kim D, Kim HJ, et al. The relation between non-alcoholic fatty liver disease and the risk of coronary heart disease in Koreans. Am J Gastroenterol 2009;104:1953–60. 28. Madamanchi NR, Vendrov A, Runge MS. Oxidative stress and vascular disease. Arterioscler Thromb Vasc Biol 2005;25:29–38. 29. Chinopoulos C, Adam-Vizi V. Calcium, mitochondria and oxidative stress in neuronal pathology. Novel aspects of an enduring theme. FEBS J 2006;273:433–50. 30. Reaven GM. Insulin resistance, the insulin resistance syndrome, and cardiovascular disease. Panminerva Med 2005;47:201–10. 31. Baskol G, Baskol M, Kocer D. Oxidative stress and antioxidant defenses in serum of patients with non-alcoholic steatohepatitis. Clin Biochem 2007;40:776–80. 32. Chang CY, Argo CK, Al-Osaimi AM, et al. Therapy of NAFLD: antioxidants and cytoprotective agents. J Clin Gastroenterol 2006;40:51–60. 33. Nobili V, Pastore A, Gaeta LM, et al. Glutathione metabolism and antioxidant enzymes in patients affected by nonalcoholic steatohepatitis. Clin Chim Acta 2005;355:105–11. 34. Park KS, Jang BK, Kwon KM, et al. Antioxidant status in nonalcoholic steatohepatitis. Korean J Hepatol 2005;11:135–43. 35. Sanyal AJ, Campbell-Sargent C, Mirshahi F, et al. Nonalcoholic steatohepatitis: association of insulin resistance and mitochondrial abnormalities. Gastroenterology 2001;120:1183–92. 36. Timlin MT, Parks EJ. Temporal pattern of de novo lipogenesis in the postprandial state in healthy men. Am J Clin Nutr 2005;81:35–42. 37. Van Steenbergen W. Niet-alcoholische steatohepatitis: een oorzaak van levercirrose bij obesiteas en diabetes mellitus. Tijdschrift Geneeskunde 1999;55:1419–24. 38. Salmenniemi U, Ruotsalainen E, Pihlajamäki J, et al. Multiple abnormalities in glucose and energy metabolism and coordinated changes in levels of adiponectin, cytokines, and adhesion molecules in subjects with metabolic syndrome. Circulation 2004;110:3842–8. 39. Pak W, Takayama F, Mine M, et al. Anti-oxidative and anti-inflammatory effects of spirulina on rat model of non-alcoholic steatohepatitis. J Clin Biochem Nutr 2012;51:227–34. 40. Merat S, Malekzadeh R, Sohrabi MR, et al. Probucol in the treatment of nonalcoholic steatohepatitis: an open-labeled study. J Clin Gastroenterol 2003;36:266–8. 41. Weiner RA. Surgical treatment of non-alcoholic steathoepatitis and non-alcoholic fatty liver disease. Dig Dis 2010;28:274–9.
Volume 348, Number 1, July 2014
Oxidative Balance in Lymphocytes From Patients With Nonalcoholic Steatohepatitis Silvia Belia, PhD, Graziana Lupattelli, MD, Eleonora Urbani, PhD, Gaetano Vaudo, MD, Anna Rita Roscini, MD, Stefano Perni, PhD and Valeria Marsili, PhD
Abstract: Oxidative stress is linked to several human diseases, including nonalcoholic steatohepatitis (NASH). In this study, lymphocytes were used as a model to study this disease. These cells offer several advantages for cellular and molecular studies such as easy accessibility, and they are easily accessible and constitute a “time-persistent” system capable of reflecting the condition of the whole organism. Lymphocytes from patients with NASH display oxidative stress features. Among the possible causes for the overproduction of reactive oxygen species in NASH lymphocytes, there might be alterations of enzymatic pathways, autooxidation of glucose and mitochondrial superoxide production, which, in turn, would lead to protein oxidative damage. Increased oxidative stress in lymphocytes from patients with NASH may result in a pro-oxidative environment, which, in turn, could modify the pathway of the enzymatic activities. The data confirm that an imbalance between pro-oxidant and antioxidant defense mechanisms may be an important factor in NASH. Key Indexing Terms: Oxidative stress; Lymphocytes; Metabolic syndrome; NAFLD; ROS. [Am J Med Sci 2014;348(1):30–36.]
N
onalcoholic fatty liver disease (NAFLD) is defined as fat accumulation in the liver exceeding 5% to 10% by weight, as microscopically determined from the percentage of fat-laden hepatocytes. The diagnosis of NAFLD implies the exclusion of the other causes of steatosis, such as viruses, alcohol, autoimmunity, toxicity, hypobetalipoproteinemia and Wilson’s disease. Nonalcoholic steatohepatitis (NASH) is the advanced form of NAFLD that progresses from simple steatosis to liver inflammation,1,2 fibrosis, cirrhosis and, possibly, hepatocarcinoma.3 NAFLD/NASH are strictly linked with obesity and insulin resistance and are frequently associated with hypertension and dyslipidemias, a constellation of abnormalities known as metabolic syndrome, and in many cases with type 2 diabetes mellitus. Metabolic syndrome is also a risk factor for NASH and for advanced fibrosis in liver tissue.4 Taken together, NAFLD and NASH affect between 9% and 33% of the general population in the developed countries. However, although NAFLD is considered a common and relatively benign liver condition, NASH is thought to potentially lead to morbidity and increased mortality, thus demanding more attention. The pathogenesis of NASH is thought to be multifactorial: the inducing factors include oxidative stress, iron deposits, overexpression of cytochrome P450E1, participation of endotoxin and tumor necrosis factor-a and mitochondrial disorders.
From the Departments of Cellular and Environmental Biology (SD, EU, SP, and Clinical and Experimental Medicine, Internal Medicine, Angiology and Atherosclerosis (GL, GV, ARR), University of Perugia, Perugia, Italy. Submitted April 24, 2013; accepted in revised form August 7, 2013. The authors have no financial or other conflicts of interest to disclose. Correspondence: Silvia Belia, PhD, Department of Cellular and Environmental Biology, University of Perugia, Via Elce di Sotto, Perugia 06123, Italy (E-mail: fi[email protected]).
Some authors have proposed the so-called “two-hit” model to describe the underlying mechanism. The “first hit” involves the accumulation of triglycerides within hepatocytes and the “second hit” regard oxidative stress that leads to inflammation, cellular injury and progressive fibrosis.5 Abnormal mitochondrial and cellular redox homeostasis has been documented in cases of steatohepatitis, which suggests that there are alterations in the signaling cascades, which would ultimately alter the function of the enzymes and proteins that are critical for mitochondrial and cellular function.6 Mitochondria play a major role in fat oxidation and energy production. They are also the main source of the reactive oxygen species (ROS) that trigger lipid peroxidation, cytokine overproduction and cell death.7 The products of ROS-induced lipid peroxidation impair the respiratory chain, leading to increased mitochondrial ROS formation, which could deplete the antioxidants and impair ROS inactivation.8 In patients with NASH, insulin resistance is the pathogenic factor that favors the accumulation of free fatty acids in the liver. Insulin resistance is also believed to induce oxidative stress by stimulating microsomal lipid peroxidases and to decrease mitochondrial beta-oxidation because of the direct effect of high insulin levels.3 To evaluate whether the pathogenesis of NASH is associated with increased levels of ROS, the following had to be ascertained: (1) significant accumulation of ROS derived from used cell metabolism; (2) definite oxygen radical damage; and (3) abnormalities in the antioxidant defenses in pathological patients. The validation of these conditions was necessary to establish the possible relationship with cascade signaling events directly correlated with the pathogenesis of the disease. Lymphocytes from peripheral blood were chosen for this study, because they can act as biosensors of the whole organism in response to the disease. These cells are not only involved in the inflammatory processes and immune responses but also contribute to homeostasis and adaptation to environmental variations and pathological conditions.9 Lymphocytes also have a remarkably long lifetime and have the capacity to keep memory of their original phenotype even after they are cultured. Most importantly, there is no evidence that highly reactive chemical species capable of interacting with biological macromolecules and altering cellular functions have any effect on lymphocytes of patients with NASH.10 For all of these reasons, lymphocytes are a robust, “time-resistant” system capable of monitoring the conditions of the entire body and keeping track of cumulative long-term stressors.9 To evaluate the involvement of oxidative stress in the pathogenesis of NASH, experiments were carried out to aim at verifying the occurrence and the level of the oxidative stress in lymphocytes derived from patients with NASH.
VM)
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MATERIALS AND METHODS Materials All media, sera, antibiotics and culture solutions were purchased from Sigma—Aldrich, Corp. (St. Louis, MO). All
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sterile culture plastics were provided by Falcon (Plymouth, United Kingdom). All other reagents were analytical grade. Patients A total of 15 overweight/obese patients (4 women and 9 men; mean age, 42 6 14 years, body mass index [BMI] 30 6 5.4 kg/m2), with a clinical diagnosis of NAFLD were included in the study. The diagnosis of NAFLD was based on the following criteria: (1) elevated aminotransferases (aspartate aminotransferase [AST] and/or alanine aminotransferase) in at least 2 determinations; (2) clinical and hematochemical exclusion of alcohol-induced or drug-induced liver disease, autoimmune or viral hepatitis and cholestatic or metabolic/genetic liver disease; and (3) sonography documenting liver steatosis. A total of 12 age-matched normal weight subjects (5 women and 8 men) from the Hospital staff were included as control group. A complete medical history and clinical examination was performed in all patients and controls. BMI was calculated using the formula: weight (in kilograms)/height (in square meters). Waist circumference was assessed by wrapping the tape around the waist midway between 2 measure points: the top of hip bone and the bottom of the ribs. Blood was drawn in the morning after a 13-hour fast and the following parameters were determined: total cholesterol, triglycerides, high-density lipoprotein-cholesterol, low-density lipoprotein-cholesterol (Friedewald’s formula), glycemia, insulinemia, AST, alkaline phosphatase, g-glutamyl transferase (GT) and apolipoprotein B. Liver Sonography Sonography of the liver was performed by a skilled ultrasonographer, unaware of clinical and laboratory patients details, using real-time l’ATL (Philips Electronics, Koninklijke, The Netherlands) equipped with a 3.5- to 5-MHz probe. Steatosis was defined sonographically by the appearance of hyperechoic liver tissue with fine, tightly packed echoes and posterior beam attenuation. Bioimpedentiometry All patients and controls underwent bioimpedentiometry (50 kHz, amplitude 50 mA; Tanita Europe B.V., Amsterdam, The Netherlands), using electrodes applied to the foot-plantar surface. This methodology allows the measurement of fat mass in kilogram and as percentage of body weight, free fat mass and water in kilogram. Vascular Ultrasound Measurement Carotid and femoral arteries were examined by highresolution B-mode ultrasonography.1 The examination was performed with a commercially available ultrasound device (ESAOTE Technos MP; ESAOTE S.p.A, Genoa, Italy) equipped with a linear multifrequency 7.5- to 12-MHz transducer. Subjects were examined in the supine position, and all measurements were obtained at enddiastole with electrocardiographic triggering. The ultrasound images were stored on a digital support and analyzed using an image processing workstation (AMS System, Gothenburg, Sweden). On a longitudinal 2-dimensional ultrasound scanning, the image of the far wall of common carotid and common femoral artery, at the prebifurcation tract, was displayed as 2 bright white lines separated by a hypoechogenic space. The intima-media thickness (IMT) of the far wall was automatically measured by the computerized system mentioned earlier (IMT common carotid and IMT common femoral). The intraobserver coefficient of variation was 1.1% (mean 6 standard deviation of the difference 0.018 6 0.031 mm), whereas interobserver values were 1.8% (0.028 6 0.032 mm). Ó 2013 Lippincott Williams & Wilkins
Flow-Mediated Vasodilation Flow-mediated vasodilation (FMV) was assessed on the brachial artery by ultrasonography. Any drug known to affect endothelial function, including nitrates, hypolipidemic drugs and aspirin, was withdrawn $1 week before the examination. Details of the procedure, which was performed according to the International Brachial Artery Task Force guidelines,3 have been reported elsewhere.4 Briefly, the measurements were performed in supine position on the nondominant arm, after 10 to 20 minutes resting in a quiet, dark room with a temperature of 22°C. The brachial artery was scanned longitudinally just above the antecubital crease using a 10-MHz probe (HDI 3500; Advanced Technology Laboratories). Diameter of the brachial artery was measured at the R-wave of the electrocardiogram, on the interface between media and adventitia of the anterior and posterior wall. Gain settings were optimized to identify the lumen and the vessel wall interfaces and were not modified during the examination. Hyperemia was induced by inflation of a pneumatic cuff (12.5 cm wide) at 230 to 250 mm Hg for 4 minutes on the most proximal portion of the upper arm. Arterial diameter measurement was repeated 45 to 60 seconds after sudden deflation of the cuff. Tracings were recorded on videotape and read by 1 investigator, who was unaware of the subject’s clinical data and temporal sequence. The average of 3 measurements of basal and posthyperemia diameter was used for the analysis. FMV was expressed as the relative increase in brachial artery diameter during hyperemia, and defined as 100 3 ([posthyperaemia diameter 2 basal diameter]/basal diameter). Blood flow was measured as arterial cross-sectional area (p 3 r2) times mean Doppler velocity corrected for angle. The intraobserver between-occasion reproducibility of FMV in our laboratory was assessed in 10 subjects examined 2 days apart. The mean 6 SD difference between the 2 examinations was 1.0% 6 1.5%.11–14 Isolation of Human Lymphocytes Peripheral venous blood samples from healthy donors and patients with NASH were collected in sodium-heparinized vacutainers. Peripheral blood lymphocytes were separated under sterile conditions on a Ficoll-Paque PLUS (Amersham, Piscataway, NY) gradient using the method by Boyum.15 Aliquots of heparinized whole blood diluted with an equal volume of Dulbecco’s phosphate-buffered saline (1:1) were gently applied to an equal volume of Ficoll-Paque PLUS in centrifuge tubes. Samples were centrifuged at 400g for 30 minutes. The resultant interface (buffy coat) was carefully aspirated from the gradient and washed twice in Dulbecco’s phosphate-buffered saline by centrifugation at 200g for 10 minutes. The subsequent pellet was resuspended in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2% L-glutamine and 1% penicillin/streptomycin. Monocytes were removed from the mononuclear fraction by adherence to Petri dishes during overnight incubation at 37°C. Purified lymphocytes were finally resuspended in complete RPMI 1640 medium (1–2 3 106 cells/mL). Cell viability was determined by Trypan blue dye exclusion. The purified lymphocytes were used for experimental analyses within 1 day from their isolation. ROS Generation Determination of Hydrogen Peroxide Production Hydrogen peroxide (H2O2) generation in lymphocytes from healthy subjects and patients with NASH was assayed using a colorimetric method involving the oxidation
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of iodide in the presence of ammonium molybdate and photometric analyses of the resulting blue starch-iodine complex performed at 570 nm.16 Briefly, human blood lymphocytes were treated with 38.5 mM HCl, 80 mM potassium iodide, 80 mM ammonium molybdate in H2SO4 and 0.38% starch. Twenty minutes after adding potassium iodide, sample absorbance was measured at 570 nm using a Titertek Multiskan MC plate reader (ICN/Flow Biochemicals, Huntsville, AL). The H2O2 concentration was estimated using a standard curve. Results are expressed as micrograms of H2O2 per 10 5 cells. Determination of O22 Release Intracellular superoxide anion (O22) production in lymphocytes from healthy subjects and patients with NASH was assayed as previously described,17 using a colorimetric method based on the reaction between nitroblue tetrazolium chloride and O22, with the consequent formation of formazan salt. Briefly, human blood lymphocytes were incubated with 1 mg/mL nitroblue tetrazolium at 37°C for 3 hours. Next, cells were centrifuged (at 500g for 10 minutes) and the pellets treated with dimethyl sulphoxide at 37°C for 20 minutes. The absorbance of formazan salt was measured at 550 nm using dimethyl sulfoxide as the blank. Scavenger Enzyme Activities The assays were performed using samples obtained from sonicated lymphocytes suspended in 20 mM Naphosphate buffer, pH 7.0, along with 1 mg/mL pepstatin, 1 mg/mL leupeptin and 100 mM phenylmethylsulphonyl fluoride as protease inhibitors and centrifuged at 100,000g for 1 hour at 4°C. Cytosol protein concentrations were measured according to the method of Lowry et al.18
Catalase activity was determined based on the decrease in absorbance because of H2O2 consumption (e 5 20.04 mM21$cm21) measured at 240 nm, according to the method previously described.19 The final reaction volume of 1 mL contained 100 mM Na-phosphate buffer, pH 7.0, 12 mM H2O2 and 70 mg of sample. Superoxide dismutase (SOD) activity was determined using the modified method of L’Abbé and Fischer.20 The final assay volume (1 mL) contained 20 mM Na2CO3, pH 10, 10 mM cytochrome c, 1 mM xanthine and xanthine oxidase. Because the xanthine oxidase activity varies, the amount used for the assay was sufficient to stimulate cytochrome c reduction at 550 nm at a rate of 0.025/min without SOD addition. SOD units were calculated on the basis of the definition that 1 unit represents the activity that inhibits cytochrome c reduction by 50%. Glutathione reductase (GR) activity was measured based on the decrease in absorbance induced by NADPH oxidation at 340 nm (e 5 26.22 mM21$cm21).21 The assay mixture contained 100 mM Na-phosphate buffer, pH 7.0, 1 mM glutathione disulphide, 60 mM NADPH and 100 mg of sample in a final volume of 1 mL. Glutathione peroxidase activity was calculated using the method of Lawrence and Burk,22 which involves the measurement of glutathione disulphide formation using a coupled enzyme system with GR. NADPH oxidation was recorded at 340 nm (e 5 26.22 mM21$cm21). Selenium dependence was determined using H2O2 as the substrate. The final reaction volume of 1 mL contained 100 mM Na-phosphate buffer, pH 7.5, 1 mM EDTA, 1 mM NaN3, 2 mM GSH, 1 U GR, 0.24 mM NADPH, 30 to 80 mg sample and 0.6 mM H2O2. Oxidative Damage of Protein Substrates The protein carbonyl content was assayed by reacting 2,4-dinitrophenylhydrazine (DNPH) and protein carbonyls.
TABLE 1. Clinical and laboratory characteristics of patients with NASH and healthy controls Parameters Controls,12 (mean 6 SD) Patients with NASH,15 (mean 6 SD) Age (yr) Waist circumference (cm), M Waist circumference (cm), F BMI (kg/m2) Fat (%) SBP (mm Hg) DBP (mm Hg) Glycemia (mg/dL) Insulin (mU/L) Total cholesterol (mg/dL) LDL-cholesterol (mg/dL) HDL-cholesterol (mg/dL) Triglycerides (mg/dL) Apoprotein B (mg/dL) Serum AST (U/L) Serum alanine aminotransferase (U/L) Serum gamma-glutamyl transferase (U/L) IMTCCR (mm) IMTCCL (mm) IMTFCR (mm) IMTFCL (mm) FMV (%)
40 89.1 87 24.07 36.8 127.08 74.92 84.54 8.74 186.92 121 47.3 71.2 63.4 19 20 28 0.83 0.83 0.75 0.76 10.76
6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6
19 7 5 2.2 3.8 16.9 8.6 9 2.2 46.9 40.4 12.8 31.5 11 7.7 8.7 11.9 0.27 0.28 0.2 0.18 1.02
42 96.4 76 30.1 39.71 126.93 74.6 96.27 13.08 217.2 141.9 41.2 208 100.3 40 66 77 1.25 0.64 0.62 0.61 7.9
6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6
14 36 44 5.4 6.5 13.7 8.4 11.1 4.4 57.9 49 11.9 110.2 29.8 15.9 53.9 43.5 1.96 0.18 0.2 0.21 1.4
P 0.06 0.02 0.02 0.01 0.18 0.61 0.83 0.55 ,0.001 0.37 0.45 0.79 ,0.001 0.005 0.01 ,0.001 0.01 0.10 0.43 0.39 0.22 0.03
FMV, flow-mediated vasodilation; HDL, high-density lipoprotein; LDL, low-density lipoprotein; SD, standard deviation; SBP, systolic blood pressure; DBP, diastolic blood pressure; IMTCCR, Intima Media Thickness Common Carotid Right; IMTCCL, Intima Media Thickness Common Carotid Left; IMTFCR, Intima Media Thickness Femoral Common Right; IMTFCL, Intima Media Thickness Femoral Common Left.
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DNPH reacts with protein carbonyls, forming a Schiff base to produce the corresponding hydrazone, which can be analyzed spectrophotometrically.23 Lymphocytes from healthy and NASH subjects were rinsed with phosphate-buffered saline to remove red blood cells and sonicated in 10 volumes of buffer containing 50 mM MES, pH 6.7 and 1 mM EDTA. The sonicated cytosolic fraction was obtained by centrifugation at 10,000g for 15 minutes at 4°C. The supernatant protein concentration was set in the range of 1 to 10 mg/mL. Eight hundred microliters DNPH or 800 mL HCl (2.5 M) was added to 200 mL protein as the blank control. Samples and blanks were left at room temperature for 1 hour in the dark and vortexed every 15 minutes. One milliliter 20% trichloroacetic acid was added to the samples. After incubation for 5 minutes on ice, samples and blanks were centrifuged at 10,000g for 10 minutes at 4°C. The resulting pellet was resuspended in 1 mL of 10% trichloroacetic acid and centrifuged at 10,000g for 10 minutes at 4°C. Next, the pellet was resuspended in 1 mL of ethanol/ethyl acetate mixture (1:1) and centrifuged at 10,000g for 10 minutes at 4°C. This step was repeated 3 times. After the final wash, protein pellets were resuspended in 500 mL of guanidine hydrochloride and centrifuged at 10,000g for 10 minutes at 4°C. The carbonyl content was determined based on supernatant absorbance at 370 nm, using a molar adsorption coefficient for DNPH of 22,000 mM21$cm21. Results are expressed as nanomoles of DNPH per milligram of proteins. Oxidative Damage of Lipidic Substrates Malondialdehyde (MDA) forms an adduct with thiobarbituric acid that can be measured by spectrophotometry. For lipid peroxidation analysis, we used the OXI-TEK TBARS Assay Kit (ZeptoMetrix Corporation; Cat. No. 0801192). We added 100 mL of sodium dodecyl sulfate to 100 mL of samples obtained by lymphocytes sonication, 2.5 mL of thiobarbituric acid buffer reagent, and then, samples were incubated at 95°C for 1 hour. The reaction was stopped by cooling samples to room temperature in an ice bath for 10 minutes. After a centrifugation at 3000 rpm for 15 minutes, the supernatant absorbance was read at 532 nm. The amount of MDA was calculated using a standard curve. Results are expressed as nanomoles of MDA per milligram of proteins. Statistical Analysis Statistical significance was calculated using the Student’s t test for unpaired data.
RESULTS Clinical and laboratory characteristics of patients with NASH and controls are summarized in Table 1. As expected, patients with NASH showed a higher BMI (P 5 0.01), waist circumference (P 5 0.02), triglycerides (P , 0.001), insulin (P , 0.001), serum AST (P , 0.01), alanine aminotransferase (P , 0.001), g-glutamyl transferase (P , 0.001) and apolipoprotein B (P 5 0.005). FMV was significantly lower in the group of patients with NASH (P 5 0.003). No significant differences emerged in IMT, both on carotid and femoral sites. ROS Generation In lymphocytes isolated from patients with NASH, we found ROS (H2O2 and O22) markedly higher than in controls. Specifically, H2O2 generation was higher in NASH lymphocytes (0.534 6 0.047 versus 0.269 6 0.025; P , 0.0001) (Figure 1A); also, a small but significant increase in the Ó 2013 Lippincott Williams & Wilkins
FIGURE 1. Reactive oxygen species generation in lymphocytes derived from patients with NASH, compared with healthy subjects. (A) Hydrogen peroxide (H2O2) production was assayed using a colorimetric method based on the oxidation of iodide in the presence of ammonium molybdate. Values are expressed as optical density (O.D.). (B) Intracellular O22 formation was assessed using the nitroblue tetrazolium reduction assay (see Methods).
concentration of O22 was observed in patients with NASH, compared with healthy subjects (0.228 6 0.073 versus 0.093 6 0.005; P 5 0.0128) (Figure 1B). Scavenger Enzyme Activity Specific activities of the main antioxidant enzymes are shown in Figure 2. Endogenous scavengers exhibited different activity in patients with NASH: a significantly lower activity of soluble catalase, a glutathione-independent H2O2 scavenging enzyme, was observed in patients with NASH when compared with the control group (20.18 6 1.267 versus 38.29 6 3.49, P 5 0.0097) (Figure 2A); on the contrary, SOD, which transforms the superoxide anion radical into oxygen and hydrogen peroxide, did not show any statistically significant difference between controls and NASH (22.89 6 1.078 versus 25.74 6 0.283; P 5 0.0628) (Figure 2B). The GR activity was significantly lower in patients with NASH compared with the controls (25.44 6 0.398 versus 56.17 6 2.388; P 5 0.0002) (Figure 2C). The
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FIGURE 2. Antioxidant enzyme activities: (A) catalase activity was determined according to the method described by Fano` et al,19 based on the decrease in absorbance because of H2O2 consumption (e 5 20.04 mM21$cm21) measured at 240 nm. (B) Superoxide dismutase (SOD) activity was determined using a modified method of L’Abbe´ and Fischer.20 SOD units were calculated using the definition of a SOD unit as the activity that inhibits the rate of cytochrome c reduction by 50%. (C) Glutathione reductase activity was measured according to the rate of decrease in absorbance induced by NADPH oxidation at 340 nm.21 (D) Glutathione peroxidase activity was calculated with the method of Lawrence and Burk,22 which involves the measurement of glutathione disulphide formation using a coupled enzyme system with glutathione reductase.
glutathione-dependent H2O2 scavenger, GPX, displays significantly higher activity in NASH than in the controls (7.93 6 0.669 versus 1.38 6 0.479; P 5 0.0013) (Figure 2D). Oxidative Damage of Protein and Lipid Substrates The oxidative damage of protein substrates, assessed from protein carbonyl levels, was significantly higher in NASH than controls (4.76 6 0.188 versus 2.71 6 0.151; P 5 0.0011) (Figure 3A). By contrast, there was no difference in the oxidative damage of lipids, because reflected by MDA levels, between control and patients with NASH (8.91 6 0.5 versus 9.73 6 0.61; P 5 0.15) (Figure 3B).
DISCUSSION The prevalence of obesity worldwide has increased dramatically worldwide over the last 3 decades. A variety of adverse health outcomes, grouped under the term of metabolic syndrome, include obesity. In this context of obesity/metabolic syndrome, the liver seems to be significantly impacted by fat deposition. It has become clear that obesity and the factors of metabolic syndrome, which encompass glucose dysregulation and dyslipidemia, are key risk factors for the development and progression of NAFLD. Multiple studies have been performed that show the relationship between insulin resistance and NAFLD and have shown that disordered insulin regulation results in multiple, altered metabolic pathways, which ultimately lead to fat deposition and inflammation in the liver.24,25 In addition, patients with metabolic syndrome are
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at high risk for developing cardiovascular disease including myocardial infarction and stroke. Furthermore, a growing body of evidence suggests that the presence of NAFLD is a risk factor for cardiovascular disease independent of the presence of the other features of metabolic syndrome factors.26,27 In this context, the production of free radicals, which form as a result of oxidative stress, is of critical importance. Oxidative stress is an imbalance between ROS production and the ability of cells, or tissues, to detoxify the reactive intermediates or to repair the resulting damage. Oxygen radicals and independent reactive radical oxygen species, and carbon, nitrogen and sulphur radicals have been linked to several human disease conditions, such as atherosclerosis, hypertension, diabetes and cancer.28 Some of the possible causes for ROS overproduction in NASH lymphocytes could be alterations of enzymatic pathways, auto-oxidation of glucose, mitochondrial superoxide production, etc.29 This state, however, produces ROS accumulation, which, in turn, leads to notable protein oxidative damage. The results of this study show increased intracellular concentrations of both O22 and H2O2 in patients with NASH: the increased concentration of superoxide anion is very dependent on mitochondrial metabolism and the structures involved in this syndrome are considered to be of the mitochondrial type.30 No significant SOD activity increase was observed in patients with NASH, and the O22 concentration was very high. The H2O2 concentration increase could, on the one hand, correspond to a reduced catalase-specific activity, or, on the Volume 348, Number 1, July 2014
Oxidative Stress in Nonalcoholic Steatohepatitis
FIGURE 3. Oxidative stress markers: (A) protein oxidative damage was measured as the protein carbonyl content. Values are derived from the reaction between DNPH and protein carbonyl. (B) Oxidative damage derived from lipid peroxidation is expressed as relative values (nmol/mg protein) of malondialdehyde.
other hand, it could be because of a combined action of GSH enzymes. In fact, even if GPX activity increases, the concomitant decreased reductase depressed activity does not supply enough substratum to the mechanism, which prevents H2O2 detoxification. As Baskol et al31 reported, in serum from patients with NASH, oxidative stress produces ROS, which induces uncontrolled lipid peroxidation, and xanthine oxidase activity is negatively correlated with SOD and GSPX activity.32 In this study, massive damage was observed in the enzymatic protein molecules involved. This damage can modify their activity resulting in structural damage. This hypothesis is confirmed by increased oxidative damage (formation of protein carbonyls) to the thiol redox state of the proteins investigated.33 Increased oxidative stress in patients with NASH may result in a pro-oxidation environment, which, in turn, could result in decreased antioxidant enzyme activities.34 The data from this study confirm that the imbalance between pro-oxidant and anti-oxidant defense mechanisms may be an important factor in the pathogenesis of NASH. The effect of ROS on the immune system was analyzed, because it responds to pathogen attacks and fights against infections. The liver is a major immune organ and functions mainly in innate immunity.35,36 Kupffer cells (KCs) in the liver can generate proinflammatory cytokines and ROS. These mediators can act locally or systemically, and the immune responses Ó 2013 Lippincott Williams & Wilkins
can lead to hepatocyte injury.37 KCs can generate hepatic ROS, which are involved in the etiology of NAFLD and in the conversion of simple hepatic steatosis to NASH. The mechanism of action of ROS in NASH formation is still unclear. There are 2 hypotheses regarding the role of the KCs in the development of NAFLD: (1) activated KCs produce ROS, which induce insulin resistance, leading to hepatocyte steatosis38; and (2) activated KCs-derived ROS cause lipid peroxidation. Products of this reaction and KC-derived tumor necrosis factor-a can result in hepatocyte injury. Failure to repair this injury can lead to the development of steatohepatitis. Increased levels of ROS in the leukocytes, activation of nuclear factor-kappa B and changes in lymphocyte surface antigen ratio (CD4+/CD8+) were observed in the NASH model rats. The administration of antioxidants, such as spirulina, reduces these variations and may decrease the inflammatory mechanisms, blocking the crosstalk between oxidative stress and inflammation and inhibiting the progression of NASH.1 Many data show that immunological processes in the liver are involved in NASH. Moreover, the percentages of CD4+ and CD8+ lymphocytes and the ratio CD4+/CD8+ provide an assessment of the immune status. This study demonstrated a real normalization of the immune imbalance after treatment with the antioxidant.39 There are many critical steps in NASH pathogenesis at which therapy can intervene. Patients with NASH must have an extremely moderate and healthy lifestyle with continuous exercise, a controlled diet and intake of antioxidant compounds such as vitamins and ursodesoxycholic acid. Many antioxidant drugs have been shown to have a positive effect in controlling enzymatic levels of the liver enzymes.40 Other studies have shown the beneficial effects of bariatric surgery,41 although controlled trials are not available yet.
CONCLUSIONS The number of patients with NASH will probably increase in the future, because this pathology is considered to be the hepatic manifestation of the metabolic syndrome, and the number of overweight individuals is destined to increase. In the future, it will be essential to understand the mechanism of progression from a nonsteatotic liver to an inflamed organ. Although many hypotheses have been proposed regarding the role of oxidative stress in the pathogenesis of NASH, much remains to be investigated. An overload of lipids mainly in the form of triglycerides is present in the liver and proinflammatory cytokines are directed toward the fatty liver. The inflamed state is aggravated by a process of lipotoxicity, which is the formation of ROS that affects the mitochondrial membranes. Impaired antiinflammatory and antioxidant defense mechanisms may be important factors in the pathogenesis of a chronic NASH. ACKNOWLEDGMENTS The authors are grateful to Professor Roberto Buonaurio and Professor Fabio Franciolini for their critical reading of the manuscript. REFERENCES 1. Adler M, Schaffer F. Fatty liver hepatitis and cirrhosis in obese patients. Am J Med 1979;67:811–16. 2. Ludwig J, Viggiano TR, McGill DB, et al. Nonalcoholic steatohepatitis: Mayo Clinic experiences with a hitherto unnamed disease. Mayo Clinic Proc 1980;55:434–8. 3. Adams LAS, Feldstein AE. Non-invasive diagnosis of nonalcoholic fatty liver and nonalcoholic steatohepatitis. J Dig Dis 2011;12:10–6.
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