Phosphorescence Oxygen Analyzer as a Measuring Tool for Cellular Bioenergetics

The “phosphorescence oxygen analyzer” and its use to monitor O2 consumption by cells and tissues are discussed in this chapter (Lo et al., 1996; Souid et al., 2003). This analytical tool assesses bioenergetics in cells undergoing apoptosis (e.g., the mitochondrial cell death pathway), in cells exposed to toxins (e.g., loss of viability) and in cells with a genetically altered energy metabolism (e.g., mitochondrial disorders) (Tacka et al., 2004a-b; Tao et al., 2007; Tao et al., 2008a). This method is applicable to suspended (e.g., Jurkat and HL-60 cells) and adherent (TU183 human oral cancer cells) cells and to fresh tissues from humans (e.g., lymphocytes, spermatozoa and tumors) and animals (e.g., liver, spleen, heart, pancreas and kidney) (Badawy et al., 2009a-b; Whyte et al., 2010; Al Shamsi et al., 2010; Al-Salam et al., 2011; Al Samri et al., 2011). The analyzer allows investigating anticancer compounds (single agents or combinations) for dosing, order of administration and exposure (Jones et al., 2009; Tao et al., 2008b; Souid et al., 2006; Goodisman et al., 2006; Tao et al., 2006a-b; Tack et al. 2004b). It can also be used to monitor reactions consuming or producing O2 (Tao et al., 2008b; Tao et al., 2009).


Relevant biological processes
The term "cellular bioenergetics" describes the biochemical processes involved in energy metabolism (energy conversion or transformation), while the term "cellular respiration" describes delivery of O 2 to the mitochondria, the breakdown of reduced metabolic fuels with passage of electrons to O 2 , and the resulting synthesis of ATP.Impaired respiration thus implies any abnormality involving cellular bioenergetics, including glycolysis.The term "apoptosis" describes cellular mechanisms responsible for initiating and executing cell death.The initiation step requires a leakage of cytochrome c from the mitochondrial intermembrane to the cytosol.In the cytosol, cytochrome c binds to the apoptotic protease activating factor-1 (Apaf-1), triggering the caspase cascade (a series of cysteine, aspartatespecific proteases).Caspase activation executes mitochondrial dysfunction (Nicholson et al., 1997).This mitochondrial perturbation involves opening the permeability transition pores (accelerating oxidations in the mitochondrial respiratory chain) and collapsing the (http://www.mccdaq.com/daq-software/universal-library.aspx).It allows direct reading from the PCI-DAS 4020/12 I/O Board (http://www.mccdaq.com/pci-dataacquisition/PCI-DAS4020-12.aspx).The software utilizes a relational database that stores experiments, pulses and pulse metadata, including slopes.Pulse identification is performed by detecting 10 phosphorescence intensities above 1.0 volt (by default).Peak identification is performed by the program which detects the highest 10% data points of a pulse and chooses the point in the group that is closest to the pulse's decay curve.Depending on the sample rate, a minimum number of data points per pulse is set and used as a cutoff to remove invalid pulses with too few data points (Shaban, 2010).Main advantages of the developed program over commercially available packages (e.g., DASYLab™ or TracerDAQ™) are provision of full control and customization of the data acquisition, storage and analysis.The choices of VB6 and Access as programming and storage environments are due to their availability, simplicity, widespread use and VB6 components that read directly from the PCI card made available by Measurement Computing.Table 1 displays identified tasks of the program.Fig. 1 shows a picture of the data acquisition system and the developed software program.Fig. 2

Instrument calibration
The instrument was calibrated with -glucose and glucose oxidase system.
The reaction contained PBS, 3 M Pd phosphor, 0.5% fat-free albumin, 50 g/mL glucose oxidase and various concentrations of -glucose.To achieve a high signal-to-nose ratio throughout the entire range of [O 2 ], the photomultiplier tube was operated at 450 volts.
Representative pulses (with exponential fits) for reactions containing PBS with 0, 125 or 500 M -glucose, 50 g/mL glucose oxidase, 3 M Pd phosphor and 0.5% fat-free bovine serum albumin are shown in Fig. 3.

Aflatoxin B1 impairs human lymphocyte respiration
Aflatoxins (most notably, aflatoxin B1) are highly carcinogenic compounds, commonly found in food contaminated by aspergillus flavus, parasiticus and penicillium species (Williams, et al., 2004;Eaton & Gallagher, 1994).These potent mycotoxins create major health problems, especially where food storage is subjected to heat and humidity.A high rate of dietary exposure is reported in Sahara Africa, China and Taiwan.For example, in eastern China (where liver cancer exceeds 1 per 10,000 population per year), an average human exposure to aflatoxins is estimated to be 2.2g/kg/day.For comparison, the exposure in the United States is about 3 orders of magnitude less (Wang et al., 1996).Biotransformation of aflatoxin B1 is critical for its activation.The parent compound undergoes oxidation by monooxygenases, especially the hepatic cytochrome P450 3A4.The active metabolite, AFB exo-8,9-epoxide, undergoes base-catalyzed rearrangement to a dialdehyde, which rapidly reacts with guanyl N7 in DNA and lysine in proteins (Johnson et al., 1996).Exposure to aflatoxin B1 has been associated with hepatocellular carcinoma (Montesano et al., 1997), mutagenesis (e.g., in the tumor suppressor gene p53) and immune suppression (Corrier, 1991).Most of the information on immunotoxicity of aflatoxin B1 is derived from animal studies (Stec et al., 2009;Reddy et al., 1987;Reddy et al., 1989;Jiang et al., 2008;reviewed in Williams, et al., 2004].In healthy humans, exposure to aflatoxin B1 is associated with lower perforin (a cytolytic protein produced by natural killer lymphocytes) expression on CD8+ T-lymphocytes (Jiang et al., 2008).A dose-related decrease in DNA synthesis in lymphocyte cultures (with and without mitogens) is found in mice exposed in vivo to aflatoxin B1 (Reddy et al., 1987).A decrease in DNA synthesis is also observed in normal splenic mouse lymphocytes cultured in vitro with >10 M aflatoxin B1; a decrease in RNA synthesis is observed at dosing >25 M and a decrease in protein synthesis at dosing >100 M (Reddy et al., 1989).
The phosphorescence oxygen analyzer is used to monitor the effects of aflatoxin B1 on human lymphocyte mitochondrial oxygen consumption.These experiments investigate whether aflatoxin B1 impairs respiration of the lymphoid tissue, an organ that is typically targeted by this potent mycotoxin.Aflatoxin B1 (1.0 mg = 3.2 micromol) was freshly dissolved in 1.0 mL dry methanol and immediately added to cell suspensions with vigorous mixing.Alternatively, aflatoxin B1 powder was directly added to the cell suspension with vigorous mixing.The concentrations were determined by the absorbance at 350 nm (10 L aflatoxin B1 stock solution or cell-free supernatant in 1.0 mL dry methanol), using an extinction coefficient of 21,500 M -1 cm -1 (Nesheim et al., 1999); the aflatoxin B1 excitation wavelength is 366 nm and the emission wavelength 455 nm.The reactions were carried out in glass vials and protected from light.PBMC (0.6 x 10 7 cells/mL) were suspended in 6.The lines are linear fit for the untreated cells (R 2 > 0.916) and exponential fit for the treated cells (R 2 > 0.936).Insert, changes in the values of k at 5-min intervals.
Fig. 6.Time-course of the effect of aflatoxin B1 on human lymphocyte respiration.
The exponential profile of O 2 consumption in the presence of aflatoxin B1 is similar to dactinomycin (Tao et al., 2006b;Tao et al., 2008a).This pattern of bioenergetic derangements could stem from progressive mitochondrial and metabolic disturbances, ranging from uncoupling oxidative phosphorylation (which accelerates O 2 consumption and rapidly depletes the metabolic fuels) to mitochondrial respiratory chain function collapse.Experimentally, these two phases are clearly distinguishable in our system (Fig. 6, insert).
Caspase activation in lymphocytes treated with aflatoxin B1 was then examined.Many of the caspases (e.g., caspase-3, -2 and -7) target the asp-glu-Val-asp (DEVD) motif and cleave at sites next to the last aspartate residue (Nicholson et al., 1997).Synthetic cell-permeable substrates, such as N-acetyl-DEVD-7-amino-4-trifluoromethyl coumarin (Ac-DEVD-AFC) and N-acetyl-DEVD-7-amino-4-methyl coumarin (Ac-DEVD-AMC) have been used to investigate caspase activities.For example, cleavage of Ac-DEVD-AFC by specific caspases releases the fluorogenic AFC; the latter can be separated on HPLC and detected by fluorescence with a great sensitivity (Tao et al., 2007).
Caspase-3 activity in lymphocytes exposed to aflatoxin B1 is shown in Fig. 7.The purpose of these experiments is to confirm caspases are activated within the time period required for inhibition of respiration.The mixtures (final volume, 0.5 mL) contained 1.5 x 10 6 cells in PBS, 10 mM glucose and 68 M Ac-DEVD-AMC (N-acetyl-asp-glu-val-asp-7-amino-4-methyl coumarin, a caspase-3 substrate) with and without 20 M zVAD-fmk (benzyloxycarbonylval-ala-DL-asp-fluoromethylketone, a pan-caspase inhibitor) (Slee et al., 1996).The suspensions were incubated at 37 o C for 2 hr without other additions (Fig. 7, left panel) or with the addition of ~100 M aflatoxin B1 (Fig. 7, right panel).At the end of the incubation period, the cells were disrupted and their supernatants were separated on HPLC and monitored by fluorescence.The results show AMC moieties (the cleavage product of Ac-DEVD-AMC) appear in the cells about 2 hr after the addition of aflatoxin B1.This 2-hr period is the same as that observed for aflatoxin B1-induced inhibition of respiration (see Fig. 5).Thus, the results suggest aflatoxin B1 impairs human lymphocyte mitochondrial function by activating caspases.
The above findings also demonstrate the lymphocyte preparation contain monooxygenases that activate aflatoxin B1.These results are consistent with previous reports (Stec et al., 2009;Rossano et al., 1999;Savel et al., 1970;Wang et al., 1999).In one study, the addition of aflatoxins B1 at concentrations up to 32 M had a minimum effect on phytohemagglutininp-stimulated human lymphocyte proliferation (Meky et al., 2001).However, an earlier study on human lymphocytes by Savel et al. (1970) showed a reduced phytohemagglutinin-pstimulated lymphocyte proliferation with 16 M aflatoxin B1.More recently, aflatoxin B1 was shown to inhibit in vitro concanavalin A-induced proliferation of pig blood lymphocytes; in 72-hr cultures, the concentration of aflatoxin B1 producing 50% inhibition (IC 50 ) was 60 nM (Stec et al., 2009).In other studies, aflatoxin G1 induced in vitro apoptosis in human lymphocytes (Wang et al., 1999;Sun et al., 2002).In summary, the data presented show human lymphocytes exposed in vitro to aflatoxin B1 exhibit impairments of cellular respiration, which could result from caspase activation.The results substantiate the potent immunosuppressive activity of aflatoxins in human.

Biocompatibility of calcined mesoporous silica particles with murine tissue bioenergetics
The in vitro system discussed in Section 8 is used to investigate the effects of two forms of calcined mesoporous silica particles (MCM41-cal and SBA15-cal) on cellular respiration of mouse tissues (Al Shamsi et al., 2010;Al-Salam et al., 2011;Tao et al., 2008c).O 2 consumption by lung, liver, kidney, spleen and pancreatic tissues was unaffected by exposure to 200 g/mL MCM41-cal or SBA15-cal for several hours.A representative experiment of pneumatocyte respiration is shown in Fig. 9.
The rate of respiration (k) was set as negative of the slope of [O2] vs. time; the values of k (in M O2 min -1 mg -1 ) are shown at the bottom of the runs.Zero minute corresponds to the addition of the particles.U, untreated; and T, treated.Normal tissue architecture and histology were confirmed by light microscopy.Intracellular accumulation of the particles in the studied tissues was evident by electron microscopy.The results show reasonable in vitro biocompatibility of the mesoporous silicas with murine tissue bioenergetics.Therefore, the measurements of respiration can be used to explore biocompatibility and viability of tissues and cells as a result of various treatments.

Liver tissue bioenergetics in concanavalin A hepatitis in mice
Concanavalin A (Con A) is a plant lectin from the seeds of Canavalia ensiformis (jack bean).This toxin serves as a polyclonal T-cell mitogen.It produces fulminant hepatitis in mice, a disease that mimics human infection with hepatitis B virus (Tiegs et al., 1992(Tiegs et al., & 1997)).The hepatic injury is typically noted within 3 hr of intravenous injection of > 1.5 mg/kg of Con A and progresses with time (Tiegs et al., 1992).Activation and recruitment of Natural Killer (NK) T-cells and other cells of the innate immune system are early events, which lead to increased secretion of various inflammatory cytokines (e.g., TNF-, IL-2, IL-10, IL-12 and IFN-) (Takeda et al., 2000;Margalit et al., 2005;Chen et al., 2010;Sass et al., 2002).This immune response targets multiple organs including the liver.Its outcome is irreversible hepatotoxicity, which includes inflammatory infiltrates and necrosis (Leist et al., 1996).The above described in vitro system is employed to assess liver tissue respiration in Con A treated C57BL/6 mice.The purpose of the work was to estimate hepatocyte bioenergetics in this well-studied hepatitis model.The mice were injected intravenously with 12 mg/kg Con A or PBS.Specimens (20 to 30 mg each) were cut from the liver of anesthetized (urethane, 100 L per 10 g body weight, using 25% solution, w/v, in 0.9% NaCl) mice using a sharp scissor (Moria Vannas Wolg Spring, cat.# ST15024-10) (Al Samri et al., 2011).The specimens were immediately immersed in ice-cold Krebs-Henseleit buffer (115 mM NaCl, 25 mM NaHCO 3 , 1.23 mM NaH 2 PO 4 , 1.2 mM Na 2 SO 4 , 5.9 mM KCl, 1.25 mM CaCl 2 , 1.18 mM MgCl 2 and 6 mM glucose, pH ~7.2), gassed with 95% O 2 :5% CO 2 .Pieces were then weighed and placed in 1-ml Pd phosphor solution (Krebes-Henseleit buffer containing 0.5% albumin and 3 M Pd phosphor) for O 2 measurement.The results are summarized in Table 3.
A representative experiment following 3-hr treatment is shown in Fig. 10.Liver tissue respiration was measured 3 hr post injection of PBS (Fig. 10, left panel) or 12 mg/kg Con A (Fig. 10, right panel).In untreated mouse, the rates of respiration at t = 0 min and t = 80 min (post tissue collection) were similar.In Con A-treated mouse, the rate of respiration at t = 0 min was high and at t = 40 min it was low.Thus, at 3-hr, Con A treatment doubled the rate of liver tissue O 2 consumption.However, respiration deteriorated in vitro in 40 min.
Minutes zero correspond to collecting the liver tissue specimens at 3 hr post injections.Two runs were done for each condition.Rates of respiration (k c , in μM O2 min -1 mg -1 ) are shown at the bottom of the runs.3) concurred with large areas of necrosis and the enhanced rate of respiration at ~3 hr post treatment (Fig. 10) concurred with inflammatory infiltrates limited to the perivascular space without any notable necrosis.The latter finding suggests a role for inflammatory mediators, such as TNF- and IL-2 (both known to peak 3 hr post Con A treatment) in modulating hepatocyte energy metabolism (Louis et al., 1997;Gottlieb et al., 2000).The mechanism for the presumed inflammationinduced increase in hepatocyte oxygen consumption could be uncoupling oxidative phosphorylation vs. up-regulating the energy metabolism.Nevertheless, for both assumptions, there is a large demand for energy supply to prevent fulminant liver necrosis.
In an in vitro experiment, liver tissue respiration was measured with and without IL-2 (added directly to the O 2 measuring vial).The rate of respiration without IL-2 was 0.21 M O 2 min -1 mg -1 and with IL-2 0.087 M O 2 min -1 mg -1 (~60% inhibition).Thus, similar to TNF-, IL-2 also inhibits cellular respiration in vitro (Gottlieb et al., 2000).

Spermatozoa respiration
The above in vitro system was also used to measure human spermatozoa respiration.O 2 concentrations in solutions containing glucose and human spermatozoa declined linearly with time.Sodium cyanide also inhibited sperm oxygen consumption, confirming the oxidations occurred in the respiratory chain.The rate of respiration (mean + SD, n = 10) was 1.0 + 0.3 M O 2 min -1 per 10 8 sperm.Immediate decline in the rate of sperm respiration was noted when toxic agents [e.g., 4-hydroperoxycyclophosphamide (4OOH-CP),  9tetrahydrocannabinol ( 9 -THC) or  8 -tetrahydrocannabinol ( 8 -THC)] were added to washed sperm or neat semen.The inhibition was concentration-dependent and irreversible (Badawy et al., 2009a-b).
The toxic effect of the cannabinoids was confirmed on isolated mitochondria from beef heart.The effect of  8 -THC on respiration of beef heart mitochondria is shown in Fig. 11.The value of k (in M O 2 min -1 ) decreased by 64% in the presence of 240 M  8 -THC.

Phosphorescence O 2 analyzer as a screening tool for disorders of impaired cellular bioenergetics
Disorders of cellular bioenergetics are challenging clinically and biochemically (Chretien and Rustin, 2003;Chretien et al., 1994;Rotig et al., 1990;Rustin et al., 1994).Their manifestations frequently overlap with numerous clinical entities.Furthermore, mutations that limit these processes in humans are incompletely identified (http://www.gen.emory.edu/mitomap.html)(Kogelnik et al., 1997).Therefore, clinicians usually rely on a laborious analysis of skin and muscle biopsies for diagnosis (Chretien and Rustin, 2003;Chretien et al., 1994;Rustin et al., 1994).As suggested by Rustin et al., laboratory evaluation of mitochondrial disorders require testing samples from multiple tissues.The authors also recommended the use of circulating lymphocytes in the initial screening (Rustin et al., 1994).These interrelations justify developing non-invasive simple screening methods that are applicable to various types of samples.Recently, Marriage et al. showed ATP synthesis in permeabilized lymphocytes is an effective screening tool for impaired oxidative phosphorylation (Marriage et al., 2003;Marriage et al., 2004).Decreased ATP synthesis in the lymphocytes was present in the 5 studied mitochondrial disorders (Marriage et al., 2003).Described herein is the use of the phosphorescence O 2 analyzer to measure lymphocyte respiration in volunteers and a patient.The measurement primarily aimed to show feasibility of using the phosphorescence O 2 analyzer to screen for clinical disorders with impaired cellular bioenergetics.Peripheral blood mononuclear cells (PBMC) were collected from healthy volunteers and patient.The rate of respiration (mean ± SD, in M O 2 per min per 10 7 cells) for adult volunteers is 2.1 ± 0.8 (n = 18), for children 2.0 ± 0.9 (n = 20), and for newborns (umbilical cord samples) 0.8 ± 0.4 (n = 18, p <0.0001).Representative experiments of the volunteers are shown in Fig. 12.For an 8-year-old patient with reduced muscle NADH dehydrogenase and pyruvate dehydrogenase activities, the rate was 0.7 ± 0.2 (n = 3) M O 2 per min per 10 7 cells.As previously noted in muscle specimens, the rate of lymphocyte mitochondrial oxygen consumption is very similar in adults and children (p =0.801) (Chretien et al., 1994).However, cord blood cells have lower rates of respiration (p <0.001).This finding could be attributed to the high number of nucleated red blood cells in the umbilical cord blood.Fresh lymphocytes were previously used as a source of tissue for measuring respiratory chain enzymes by polarography (Clark-type O 2 electrode) and spectroscopy (Chretien and Rustin, 2003;Chretien et al., 1994;Rustin et al., 1994).Rotig et al. reported a rate (mean + SD, n=15) of 3.5 + 0.5 nmol O 2 per min per 10 7 cells (Rotig et al., 1990).Hedeskov and Esmann reported a rate of 2.0 + 0.07 nmol O 2 per min per 10 7 for cell concentrations >4 x 10 7 per mL and higher rates for less concentrated cells (Hedeskov and Esmann, 1966).Pachman reported rate of 1.0 + 0.2 nmol O 2 per min per 10 7 equine lymphocytes (Pachman, 1967).Clinical presentations of entities with impaired cellular bioenergetics vary markedly.Their manifestations may include progressive neuromuscular defects (e.g., psychomotor retardation and hypotonia), heart muscle involvement and encephalopathy.One typical example is Leigh syndrome, which results from an isolated mitochondrial complex I deficiency (Benit et al., 2004).This clinical heterogeneity stems from various mechanisms, including tissue-specific of nuclear-encoded isoforms of the respiratory chain and existence of normal and mutated mtDNA in the same organ (mtDNA heteroplasmy) (Rustin et al., 1994).Therefore, as suggested by Rustin et al., the biochemical analysis should not be limited to skeletal muscle and skin tissues (Rustin et al., 1994).In one study, 42 patients with respiratory chain defects were investigated.The results showed that 50% of the patients had deficiencies in skeletal muscles and lymphocytes, 45% in skeletal muscles only, and 5% in lymphocytes only (Chretien et al., 1994).Patients with Pearson's syndrome on the other hand consistently express defects in the lymphocyte (Rotig et al., 1990).

Conclusions
A novel in vitro system that allows monitoring of cellular respiration over several hours is described.The method has numerous biological applications, including studying mitochondrial dysfunction during apoptosis or toxic exposure.It also allows screening for metabolic disorders in patients.The procedure is sensitive and reproducible.It is applicable to cells in suspension, adherent cells and various organs, including the heart muscle, liver, spleen, pancreas and kidney.
shows a reaction vial.Experiment identification (title, date, time and sample rate) Reading directly from the PCI card at the fastest possible rate Distinguishing pulse data from non-pulse data Allowing a fuzzy detection of the pulse peak Calculating the exponential decay rate (1/) and lifetime () of each pulse Storing each pulse data points, along with the peak, decay and lifetime values Viewing a representative pulse every 10 sec Viewing decay rates (1/) in a second graph Ability to pause and place a marker with a note Ability to remove erroneous (incomplete) pulses and adjust peak values if necessaryAbility to copy pulse or slope data to clipboard for further analysis Ability to access a previous experiment, review a pulse with its metadata, markers and associated notes Table1.Identified tasks of the customized software program for data acquisition, storage and analysisThe components (panel A, left to right) are circulating water bath, power supply for the mixer, sample chamber (panel B) attached to PMT, high voltage power supply for PMT, computer with PCI-DAS board, and monitor with developed software running.The PMT is connected to PCI-DAS board on the back of the computer.The developed software program interface is shown in panel C.

Fig. 1 .
Fig. 1.The data acquisition system and developed software program.

Fig. 4 .
Fig. 4. Calibration with -glucose plus glucose oxidase 0 mL PBS, 10 mM glucose, 3 M Pd phosphor and 0.5% fat-free bovine serum albumin.The mixture was divided into 2 equal aliquots.Methanol (25 L per mL, Fig. 5, left panel) or aflatoxin B1 (25 M, Fig. 5, right panel) was then added and the incubation continued at 37 o C (open to air with gentle stirring).At t = 10 and 110 min, 1.0 mL of each mixture was simultaneously placed in the instruments for O 2 measurement.The rate of respiration (k, in M O 2 min -1 ) for t = 10 to 78 min for the methanol-treated cells was 2.0 and for the aflatoxin B1-treated cells 2.1.The values of k for t = 110 to 174 were 2.2 and 1.2, respectively (corresponding to 45% inhibition of lymphocyte respiration).PBMC were incubated at 37 o C with 25 μL per mL methanol (left panel) or 25 μM aflatoxin B1 (right panel).Minute zero corresponds to the addition of aflatoxin B1.At t = 10 and t = 110 min, 1.0 mL of each mixture was simultaneously placed in the instruments for O2 measurement.Rates of respiration (k) were calculated from the best-fit linear curves.Additions of 5.0 mM NaCN and 50 g/mL glucose oxidase are shown.
The reactions contained 1.5 x 10 6 cells in PBS plus 10 mM glucose and 68 M Ac-DEVD-AMC with and without 20 μM zVAD-fmk.The suspensions were incubated at 37 o C for 2 hr without other additions (left panel) or with the addition of ~100 M aflatoxin (right panel).At the end of the incubation period, the cells were disrupted and their supernatants were separated on HPLC and monitored by fluorescence.The retention time for Ac-DEVD-AMC was ~2.4 min and for the released AMC ~8.7 min.

Fig. 10 .
Fig. 10.Representative experiment for liver tissue respiration in C57BL/6 mice 3 hr post injection of PBS (left panel) or 12 mg/kg Con A (right panel) Thus, Con A treatment produced a concurrent impairment of hepatocyte respiration.The lower rate of respiration at 12 hr post treatment (Table3) concurred with large areas of necrosis and the enhanced rate of respiration at ~3 hr post treatment (Fig.10) concurred with inflammatory infiltrates limited to the perivascular space without any notable necrosis.The latter finding suggests a role for inflammatory mediators, such as TNF- and IL-2 (both known to peak 3 hr post Con A treatment) in modulating hepatocyte energy metabolism(Louis et al., 1997;Gottlieb et al., 2000).The mechanism for the presumed inflammationinduced increase in hepatocyte oxygen consumption could be uncoupling oxidative phosphorylation vs. up-regulating the energy metabolism.Nevertheless, for both assumptions, there is a large demand for energy supply to prevent fulminant liver necrosis.In an in vitro experiment, liver tissue respiration was measured with and without IL-2 (added directly to the O 2 measuring vial).The rate of respiration without IL-2 was 0.21 M O 2 min -1 mg -1 and with IL-2 0.087 M O 2 min -1 mg -1 (~60% inhibition).Thus, similar to TNF-, IL-2 also inhibits cellular respiration in vitro(Gottlieb et al., 2000).

Fig. 12 .
Fig. 12. Left panel: Lymphocytes (1.0 x 10 7 cells/mL) were collected from a 9-year-old girl; the rate of respiration was 2.8 M O 2 per min per 10 7 cells (R 2 >0.942).Right panel: Lymphocytes (15 x 10 7 cells/mL) were collected from umbilical cord; the rate of respiration was 8.0 M O 2 per min (R 2 >0.934), or 0.5 M O 2 per min per 10 7 cells.The additions of 5.0 mM NaCN and 50 g/mL glucose oxidase are shown.