Reagents
Atorvastatin was purchased from Selleck Chemicals (Houston, TX, USA). Pd(II) complex of meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin (Pd phosphor) was purchased from Porphyrin Products (Logan, UT). A lyophilized powder of caspase inhibitor I [N-benzyloxycarbonyl-val-ala-asp(O-methyl)-fluoromethylketone, zVAD-fmk, m.w. = 467.5, a pan-caspase inhibitor] was purchased from Calbiochem (La Jolla, CA). Ac-DEVD-AMC (N-acetyl-asp-glu-val-asp-7- amino-4-methylcoumarin; m.w. = 675.64; caspase-3 substrate) was purchased from Axxora LLC (San Diego, CA). Complete® protease inhibitor cocktail was purchased from Roche Applied Science (Indianapolis, IN). Glucose (anhydrous), endotoxin- and fatty acid-free bovine serum albumin and remaining reagents were bought from Sigma-Aldrich (St. Louis, MO).
The pan-caspase inhibitor (zVAD-fmk) solution (2.14 mM) was made by dissolving 1.0 mg in 1.0 mL dimethyl sulfoxide and stored at -20°C. Ac-DEVD-AMC solution (7.4 mM) was made by dissolving 5.0 mg in 1.0 mL dimethyl sulfoxide and stored at -20°C. Pd phosphor solution (2.5 mg/mL = 2 mM) was prepared in dH2O and stored in aliquots at -20°C. NaCN solution (1.0 M) was prepared in dH2O; the pH was adjusted to ~7.0 with 12 N HCl and stored at -20°C. Glucose oxidase, 10 mg/mL in dH2O, was stored at -20°C. One tablet of Complete® protease inhibitor cocktail was dissolved in 1.0 mL Water-For-Injection and stored at -20°C.
Mice
Male Taylor Outbred (TO, age: 9-10 weeks, weight: 30-35 g) and C57Bl/6 (age: 9-10 weeks, weight: 20-22 g) mice were maintained at an animal facility that was in compliance with NIH guidelines (http://grants.nih.gov/grants/olaw/references/phspol.htm). The mice were purchased from the Jackson Laboratory (Bar Harbor, ME). All mice were housed in rooms maintained at 22°C with ~60% relative humidity and a 12-hr light/dark cycle. All mice had ad libitum access to standard rodent chow and filtered water. All protocols here received approval from the Animal Ethics Committee-United Arab Emirates University-College of Medicine and Health Sciences.
Liver tissue
Mice were anesthetized by sevoflurane inhalation (100 μL per 10 g body weight). Liver specimens (~20 to 30 mg) were collected using a 4-mm human skin biopsy punch (Miltex GmbH, Germany) and immediately immersed in 50 mL ice-cold modified Krebs-Henseleit (KH) buffer (115 mM NaCl, 25 mM NaHCO3, 1.23 mM NaH2PO4, 1.2 mM Na2SO4, 5.9 mM KCl, 1.0 mM EDTA, 1.18 mM MgCl2, 10 mM glucose, and 0.5 μL/mL Complete® protease inhibitor cocktail, pH 7.5) continuously gassed with 95% O2: 5% CO2[18].
The samples were then incubated in vitro at 37°C in 50 mL in KH buffer (115 mM NaCl, 25 mM NaHCO3, 1.23 mM NaH2PO4, 1.2 mM Na2SO4, 5.9 mM KCl, 1.25 mM CaCl2, 1.18 mM MgCl2, and 10 mM glucose, pH 7.5) supplemented with 0.5 μL/mL Complete® protease inhibitor cocktail and continuously gassed with 95% O2: 5% CO2. For the O2 measurement, specimens were placed in 1.0 mL KH buffer (air-saturated) containing 0.5% fatty acids-free bovine albumin and 3 μM Pd phosphor. Specimens were also processed for measuring caspase activity, urea synthesis and histology as described below.
Oxygen measurement
Phosphorescence oxygen analyzer was used to monitor O2 consumption by liver specimens [18, 19]. O2 detection was performed with the aid of Pd phosphor that had absorption maximum at 625 nm and phosphorescence maximum at 800 nm. Samples were exposed to light flashes (600 per min) from a pulsed light-emitting diode array with peak output at 625 nm (OTL630A-5-10-66-E, Opto Technology, Inc., Wheeling, IL). Emitted phosphorescent light was detected by a Hamamatsu photomultiplier tube (928) after first passing it through a wide-band interference filter centered at 800 nm. The amplified phosphorescence decay was digitized at 1.0 MHz by a 20-MHz A/D converter (Computer Boards, Inc., Mansfield, MA).
A program was developed using Microsoft Visual Basic 6, Microsoft Access Database 2007, and Universal Library components (Universal Library for Measurements Computing Devices; http://www.mccdaq.com/daq-software/universal-library.aspx). It allowed direct reading from the PCI-DAS 4020/12 I/O Board (PCI-DAS 4020/12 I/O Board; http://www.mccdaq.com/pci-data-acquisition/PCI-DAS4020-12.aspx). The pulse detection was accomplished by searching for 10 phosphorescence intensities >1.0 volt (by default). Peak detection was accomplished by searching for the highest 10 data points of a pulse and choosing the data point closest to the pulse decay curve [20].
The phosphorescence decay rate (1/τ) was characterized by a single exponential; I = Ae-t/τ, where I = Pd phosphor phosphorescence intensity. The values of 1/τ were linear with dissolved O2: 1/τ = 1/τo + k
q
[O2], where 1/τ = the phosphorescence decay rate in the presence of O2, 1/τo = the phosphorescence decay rate in the absence of O2, and k
q = the second-order O2 quenching rate constant in s-1 • μM-1.
Respiration was measured at 37°C in 1-mL sealed vials. Mixing was with the aid of parylene-coated stirring bars. In vials sealed from air, [O2] decreased linearly with time, indicating the kinetics of mitochondrial O2 consumption was zero-order. The rate of respiration (k, in μM O2 min-1) was thus the negative of the slope d[O2]/dt. NaCN inhibited respiration, confirming O2 was consumed in the mitochondrial respiratory chain.
The calibration reaction contained PBS with 3 μM Pd phosphor, 0.5% fat-free albumin, 50 μg/mL glucose oxidase and various concentrations of β-glucose. [O2] was calculated using, 1/τ = 1/τo + k
q
[O2] [21]. Rates of cellular respiration were normalized per mg of liver tissue (i.e., expressed as μM O2 consumed per min per mg liver tissue).
ATP content
Liver fragments were homogenized in ice-cold 2% trichloroacetic acid for 2 min and neutralized with 100 mM Tris-acetate, 2 mM EDTA, pH 7.75. The supernatant was collected by centrifugation (1000 × g at 4°C for 5 min) and stored at -20°C until analysis. The pH of samples was adjusted to 7.75 immediately before ATP determination. ATP concentration was measured using the Enliten ATP Assay System (Bioluminescence Detection Kit, Promega, Madison, WI). Briefly, 2.5 μL of the acid-soluble supernatant was added to 25 μL of the luciferin/luciferase reagent. The luminescence intensity was measured at 25°C using Glomax Luminometer (Promega, Madison, WI). The ATP standard curve was linear from 10 pM to 100 nM (R
2 >0.9999).
Intracellular caspase activity
Liver specimens were incubated at 37°C in oxygenated KH buffer containing 37 μM Ac-DEVD-AMC with and without 32 μM zVAD-fmk (final volume, 0.5 mL). The tissue was then disrupted by vigorous homogenization and 10 passages through a 27-G needle. The Ac-DEVD-AMC cleavage reaction was quenched with tissue disruption, mainly because caspases became inactive due to dilution. The supernatant was collected by centrifugation (16,300 g for 90 min) through a Microcentrifuge Filter (nominal molecular weight limit = 10,000 Dalton, Sigma©), separated on HPLC, and analyzed for the free fluorogenic AMC moiety. The elution time for AMC was about 5.0 min.
HPLC
The analysis was performed on a Waters 1525 reversed-phase HPLC system (Spectra Lab Scientific Inc, Alexandria, VA) that consisted of manual injector, pump and fluorescence detector. The excitation wavelength used was 380 nm and the emission wavelength 460 nm. Solvents A and B were HPLC-grade CH3OH: dH2O (1:1; isocratic). The Ultrasphere IP column (4.6 × 250 mm, Beckman) was operated at 25°C at 1.0 mL/min. The run time was 20 min. The injection volume was 50 μL.
Urea synthesis
Liver specimens were incubated at 37°C in 50 mL KH buffer (continuously gassed with 95% O2: 5% CO2) with and without 1.0 μM atorvastatin for up to 6 h. Specimens were then removed from the incubation solution every 60 min and placed in 1.0 mL KH buffer supplemented with 10 mM NH4Cl and 2.5 mM ornithine. The reactions were allowed to continue at 37°C for 50 min with continuous gassing as above. At the end of the incubation period, the solutions were analyzed for urea as described [22]. Blood urea nitrogen (BUN, mmol/L) was measured using standard laboratory methods with an LX20 multiple automated analyzer (Beckman Coulter, CA, USA). For conversion, BUN (mg/dL) = BUN (mmol/L) ÷ 0.357; Urea (mg/dL) = BUN (mg/dL) × 2.14.
Histology
Liver samples were fixed in 10% neutral formalin, dehydrated in increasing concentrations of ethanol, cleared with xylene and embedded in paraffin. Four-micrometer sections were prepared from paraffin blocks and stained with hematoxylin and eosin.
Statistical analysis
Data were analyzed using SPSS statistical package (version 19). The nonparametric test (2 independent variables) Mann-Whitney was used to compare treated and untreated samples. Respiration rates (k
c
, in μM O2 min-1 mg-1), cellular ATP content (pmol mg-1), AMC peak areas (arbitrary unit mg-1) and urea (mg/dL per mg liver tissue) for untreated samples were compared with those for treated samples.