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Clinical Infection & Immunity

Original Article

Volume 3, Number 2, June 2018, pages 60-64

The Mitochondrial Function of Patient’s Chronic Hepatitis C Over Respiratory States Ranging

The hepatitis C virus (HCV) affects 3% of the world population and causes a clinically important disease [1, 2]. Hepatitis C virus is recognized as a major factor in fibrosis and cirrhosis development. Parenchymal damage of cell membranes may lead to metabolic disorders, which plays a major role in liver fibrosis formation in hepatitis C [2, 3]. The research dating several decades has usually been carried out in functional studies of isolated mitochondria in the absence of adenosine triphosphate (ADP) [1]. In many cases, investigators have used the data to calculate parameters including the respiratory control ratio or the amount of ADP consumed per amount of oxygen utilized [1]. Such studies have been widely applied to describe mitochondrial function as affected by a myriad of physiological or pathophysiological states.

The components for metabolomic function of hepatocytes, including mitochondrial function, respiratory states and endogenic intoxication, have been assessed in the past by different methods designed to regenerate ADP for phosphorylation. These include the use of creatine, creatine kinase [2-4], ATPase with excess ATP [2, 5-7] and ratio NAD⁺/NADH₂, glucose/hexokinase [4, 8].

Most of these studies were directed at liver mitochondria and HCV establishes a chronic infection in the face of an active immune response and the host oxidative defense. However, little is known about how the virus can survive in a highly oxidative environment given that oxidative stress is such a prominent clinical feature associated with hepatitis C infection [6-12]. Adaptation to oxidative stress is key to virus survival.

The aim here of using ADP recycling technology is to assess erythrocyte mitochondrial function of patient’s chronic hepatitis C over respiratory states ranging NAD⁺/NADH₂.

The 62 HCV⁺ patients and 24 healthy controls were enrolled in the present cross-sectional study. The patients were selected on the basis of their stable clinical condition over the past 3 months.

The study protocol was carried out in accordance with the Helsinki Declaration as revised in 1989. All subjects were informed about the study and the written consent was obtained from each one.

HCV infection was diagnosed by the positivity of anti-HCV and HCV-RNA for at least 6 months of period.

History of alcohol abuse, smoking habit, pregnancy, and antioxidant use, fish-oil or iron supplement in the previous month, receiving antiviral and/or interferon therapy, uncontrolled elevated blood pressure, serum total bilirubin level higher than 2 mg/dL, concomitant chronic hepatitis B or other well-known liver diseases such as metabolic or autoimmune disorders and various infectious states of the liver, human immune deficiency virus infection, diabetes mellitus, chronic respiratory insufficiency, rheumatoid arthritis, cirrhosis or malignant tumor.

Anti-HCV was assayed by microparticle ELISA method (Anti-HCV ELISA Kit; Diagnostic Automation/Cortez Diagnostics, Inc., USA). HCV-RNA was determined using real-time polymerase chain reaction (RT-PCR) method (RoboGene^® HCV RNA Quantification Kit; Analytik Jena) in BioRad ICycler. Upper and lower detection limit (68 IU/mL).

The erythrocytes were washed twice in phosphate-buffered saline (145 mM NaCl, 5 mM NaPi and 1 mM EDTA, pH 7.4) and white cells were removed by filtration through cellulose [9]. Total erythrocyte mitochondria were prepared by differential centrifugation and purification on a Percoll [10]. Mitochondrial integrity was assessed by cytochrome C release using a commercial kit (Cytochrome C Oxidase Assay Kit; Sigma-Aldrich, St Louis, MO, USA) indicating a mean of 96% intact mitochondria [11].

The ADP/ATP ratio assay kit provides a simple and direct procedure for measuring ADP and ATP levels in cells for the screening of apoptosis, necrosis and cell proliferation. The assay involves two steps. In the first step, the working reagent lyses cells to release ATP and ADP. In the presence of luciferase, ATP immediately reacts with the substrate D-luciferin to produce light. The light intensity is a direct measure of the intracellular ATP concentration.

Luciferase: ATP + D-Luciferin + O₂ → oxyluciferin + AMP + PPi + CO₂ + light

In the second step, the ADP is converted to ATP through an enzyme reaction. This newly formed ATP then reacts with the D-luciferin as in the first step. The second light intensity measured represents the total ADP and ATP concentration in the sample.

Intrinsic NADH fluorescence was monitored in isolated mitochondria as a marker of the mitochondrial NADH redox state. Isolated mitochondria were added to individual wells of 96-well black round-bottom microplates at a final concentrations of 0.1 mg/mL in 60 µL of respiration medium containing 105 mM KCl, 10 mM NaCl, 5 mM Na₂HPO₄, 2 mM MgCl₂, 10 mM HEPES (pH 7.2), 1 mM EGTA, 0.2% defatted BSA, 5 units/mL hexokinase and 5 mM 2-deoxyglucose. Respiratory substrates and ADP were present as indicated. Fluorescence was monitored in Dynex/Dynatech Microplate Luminometer (MTX Lab Systems, USA) at 37 °C. Measurements were taken emission wavelengths of 340 nm, respectively. NADH concentration (in µmol) in the sample is calculated according to formula: X = (Δ340 × V)/6.22, where Δ340 represents change in reaction mixture optical density and V represents volume of sample in mL. NADH molar absorption coefficient at 340 nm is 6.22 × 10³/mol/cm.

H₂O₂ production was assessed simultaneously with ATP production using methods described by Yu [10]. Fluorescence was measured and quantification carried [13]. A total of 0.5 mL of isolated cells in HBSS (about 5×10⁶ cells/mL) was added to the tubes containing 1.5 mM cytochrome c or ferricytochrome c. The formation of H₂O₂ in red blood cells (RBCs) was followed by measuring the oxidation of acetylated ferrocytochrome c catalyzed by cytochrome c peroxidase at room temperature. This method was based on the measurement for the oxidation rate of reduced cytochrome c H₂O₂, when the H₂O₂ generation rate was higher than 50 nM/min. While the formation of superoxide in the animal cells was monitored by the reduction of acetylated ferricytochrome c.

The O₂ dissociation curve was determined in the last aliquot of red cell suspension with a micropbotometric reaction apparatus equilibrating a microsample of sample with gas mixtures of known PO₂ and PCO₂ and measuring blood O₂ saturation (SO₂) microphotometrically [13].

Mitochondria (0.05 mg/mL) were incubated at 37 °C in 2 mL of ionic respiratory buffer (105 mM KCl, 10 mM NaCl, 5 mM Na₂HPO₄, 2 mM MgCl₂, 10 mM HEPES, pH 7.2, 1 mM EGTA, 0.2% defatted BSA) with 5 U/ml hexokinase (Worthington Biochemical) and 5 mM 2-deoxyglucose. A tetraphenylphosphonium standard curve was performed in each run by adding tetraphenylphosphonium chloride at concentrations of 0.25, 0.5, 0.75 and 1 µM prior to the addition of mitochondria to the chamber.

An independent (unpaired) Student’s t-test (two-tailed) was chosen to test the significance of differences among means of small “n” sample sets.

There were no statistically significant differences between the groups with respect to age and gender (P > 0.05). No correlation was observed between alanine aminotransferase (ALT) and HCV-RNA level in HCV-infection patients (P > 0.05).

HCV infection induces endogenic intoxication and oxidative stress salters which cause mitochondrial dysfunction and hepatocytes damage. The HCV-infected cells displayed distinct and mitochondrial injury of fragmented mitochondria, such as swollen mitochondria devoid of mitochondrial cristae. The typical mitochondria have been displayed of tubular which displayed a network indicative of normal healthy cells (Fig. 1).

Click for large image

Figure 1. The mitochondrial HCV-infected and healthy cells.

The appearance of membrane-structural enzymes in higher values in patients serum may indicate and reflect the development of membrane pathology in which dysfunction of nuclear-cytoplasmic interactions is formed, intracellular metabolism and bioenergetics processes are disturbed and it is important in the pathogenesis of hepatitis.

We observed a progressive increase in O₂ flux with incremental additions of clamped ADP in group 2 - health. However, O₂ correlations (P < 0.05) exceeded in group 1 - HCV-infected patients in comparison with group 2 content.

For respiration group 1 - HCV-infected patients on succinate without rotenone, O₂ flux initially increased, which was followed by markedly decreased respiration as ADP was further increased.

The total levels of ATP and ADP secreted the patients with HCV was 2.25 ± 0.35 and 3.40 ± 0.45 nmol. The ratio of ATP to ADP, secreted from platelets upon activation, was 1:1.60, respectively (Fig. 2a). Mitochondrial dynamics and quality control are tightly linked to cellular metabolic alterations and ATP levels. To investigate whether inhibition in HCV secretion is a result of reduction in cellular ATP levels, we determined the total ATP levels and rate of glycolysis, an alternative mode of ATP generation.

Click for large image

Figure 2. Inhibition of mitochondrial fission and mitochondrial function energized. (a) The O2 with incremental additions of clamped ADP and ratio ATP/ADP; (b) lactate/pyruvate ratio, NADH/NAD+ ratio.

To detect bioenergetics changes we examined the ratio of pyruvic acid and lactate as markers of carbohydrate metabolism oxidative stage (the ratio of aerobic and anaerobic phases), and NAD⁺ and NADH₂ levels as mandatory participants of oxidation-reduction reactions and regulators of cell metabolism. Decreased NADH₂ index (0.002 ± 0.0001 mmol/L) was determined in comparison with control group (0.01 ± 0.0005 mmol/L). The NAD⁺ concentration (0.494 ± 0.03 mmol/L) was significantly (P < 0.05) increased in patients HCV infection in comparison with normal content, respectively (Fig. 2b). The NADH fluorescence did not change at low ADP in patients HCV infection, decreased of rotenone. The production of reactive oxygen species (ROS) was very high under HCV infections. An increased content of oxidized nicotinamide coenzymes was detected in patients with chronic hepatitis C.

Study of lactate and pyruvate parameters has found the following. In patients of group 1, lactate indexes exceeded the parameters of control group and amounted to 2.12 ± 0.23 and 1.89 ± 0.45 mmol/L in comparison with control value (1.56 ± 0.235 mmol/L). Pyruvate serum indexes were significantly lower than in the control group (0.056 ± 0.011 mmol/L) and composed accordingly for patients HCV-infections 0.031 ± 0.012 mmol/L, respectively (Fig. 2b). Pyruvate, which re-oxidizes cytosolic NADH to NAD⁺, completely abrogated the increases in HCV replication.

Increase in the ratio of NAD⁺/NADH₂ reduces the activity of NAD⁺-dependent enzymes in the cytosol and mitochondria. Restoration of dehydroxyacetonphosphate which is an intermediate metabolite of glycolysis and glyconeogenesis leads to inhibition of the last. Increased concentration of NADH₂ compared with NAD⁺ slows lactate oxidation and increases the ratio of lactate/pyruvate resulting in even more slowdown of glyconeogenesis. Lactate concentration increases in the blood.

Pyruvate oxidative decarboxylation is accompanied by formation of NADH, which brings electrons to the respiratory chain and provides ATP synthesis. As the ratio of NAD⁺/NADH₂ is relatively constant in cells so the increase in NADH concentration reduces the rate of pyruvate decarboxylation. Therefore, NAD⁺/NADH₂ ratio change is an important indicator which reflects the energy needs of cells that regulate the rate of pyruvate oxidation decarboxylation. Catalytic activity of pyruvate dehydrogenase complex decreases when cells have enough fatty acids that we observed in our study.

Mitochondrial fission is the key determinant of mitochondrial quality control, and HCV modulates these key processes in the adaptation to cellular physiological perturbations associated with infection to promote viral persistence. Mitochondrial fission is not invariably associated with cell death but can also protect cells from death induced by oxidative stress and Ca²⁺-dependent apoptotic stimuli. The mechanism by which ROS suppresses HCV replication is still not completely clear, but it is likely to involve calcium and the dissociation of HCV replication complex from the membranes. Detailed understanding of the mechanism by which ROS suppresses HCV replication and how acetaldehyde, NADH, acetyl-CoA and ROS affect HCV will require additional studies.

Conflict of Interest

This article does not have of conflict of interest.

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