\* *<sup>P</sup>*-value = 0.005

Table 3. Liver tissue respiration in Con A treated C57BL/6 mice. Mice were injected with Con A or PBS. Liver specimens were collected 12 hr post injection.

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 O2 consumption. However, respiration deteriorated *in vitro* in 40 min.

tissue bioenergetics. Therefore, the measurements of respiration can be used to explore

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 & 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 NaHCO3, 1.23 mM NaH2PO4, 1.2 mM Na2SO4, 5.9 mM KCl, 1.25 mM CaCl2, 1.18 mM MgCl2 and 6 mM glucose, pH ~7.2), gassed with 95% O2:5% CO2. Pieces were then weighed and placed in 1-ml Pd phosphor solution (Krebes-Henseleit buffer containing 0.5% albumin and

biocompatibility and viability of tissues and cells as a result of various treatments.

**10. Liver tissue bioenergetics in concanavalin A hepatitis in mice** 

3 M Pd phosphor) for O2 measurement. The results are summarized in Table 3.

\* *<sup>P</sup>*-value = 0.005

of liver tissue O2 consumption. However, respiration deteriorated *in vitro* in 40 min.

Con A or PBS. Liver specimens were collected 12 hr post injection.

Table 3. Liver tissue respiration in Con A treated C57BL/6 mice. Mice were injected with

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

*kc* **(M O2 min-1 mg-1)**  mean + SD (n) range

PBS 0.26 + 0.04 (5) 0.22 – 0.32

12 mg/kg Con A 0.18 + 0.03 (5)\* 0.13 – 0.20

**Strain Treatment** 

C57Bl/6

Minutes zero correspond to collecting the liver tissue specimens at 3 hr post injections. Two runs were done for each condition. Rates of respiration (*kc*, in μM O2 min-1 mg-1) are shown at the bottom of the runs.

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 (Table 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 O2 measuring vial). The rate of respiration without IL-2 was 0.21 M O2 min-1 mg-1 and with IL-2 0.087 M O2 min-1 mg-1 (~60% inhibition). Thus, similar to TNF- , IL-2 also inhibits cellular respiration *in vitro* (Gottlieb et al., 2000).

#### **11. Spermatozoa respiration**

The above *in vitro* system was also used to measure human spermatozoa respiration. O2 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 O2 min-1 per 108 sperm. Immediate decline in the rate of sperm respiration was noted when toxic agents [e.g., 4-hydroperoxycyclophosphamide (4OOH-CP), 9 tetrahydrocannabinol (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).

Phosphorescence Oxygen Analyzer as a Measuring Tool for Cellular Bioenergetics 251

Fig. 12. Left panel: Lymphocytes (1.0 x 107 cells/mL) were collected from a 9-year-old girl;

Lymphocytes (15 x 107 cells/mL) were collected from umbilical cord; the rate of respiration was 8.0 M O2 per min (*R*2 >0.934), or 0.5 M O2 per min per 107 cells. The additions of 5.0

For an 8-year-old patient with reduced muscle NADH dehydrogenase and pyruvate

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 O2 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 O2 per min per 107 cells (Rotig et al., 1990). Hedeskov and Esmann reported a rate of 2.0 + 0.07 nmol O2 per min per 107 for cell concentrations >4 x 107 per mL and higher rates for less concentrated cells (Hedeskov and Esmann, 1966). Pachman reported rate of 1.0 + 0.2 nmol O2 per min per 107 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

the rate of respiration was 2.8 M O2 per min per 107 cells (*R*2 >0.942). Right panel:

dehydrogenase activities, the rate was 0.7 ± 0.2 (n = 3) M O2 per min per 107 cells.

hand consistently express defects in the lymphocyte (Rotig et al., 1990).

mM NaCN and 50 g/mL glucose oxidase are shown.

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 O2 min-1) decreased by 64% in the presence of 240 M 8-THC.

Fig. 11. 8-THC added to isolated mitochondria from beef heart.

## **12. Phosphorescence O2 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 O2 analyzer to measure lymphocyte respiration in volunteers and a patient. The measurement primarily aimed to show feasibility of using the phosphorescence O2 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 O2 per min per 107 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.

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 O2 min-1) decreased by 64% in the presence of 240 M 8-THC.

Fig. 11. 8-THC added to isolated mitochondria from beef heart.

**impaired cellular bioenergetics** 

(Marriage et al., 2003).

of the volunteers are shown in Fig. 12.

**12. Phosphorescence O2 analyzer as a screening tool for disorders of** 

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

Described herein is the use of the phosphorescence O2 analyzer to measure lymphocyte respiration in volunteers and a patient. The measurement primarily aimed to show feasibility of using the phosphorescence O2 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 O2 per min per 107 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

Fig. 12. Left panel: Lymphocytes (1.0 x 107 cells/mL) were collected from a 9-year-old girl; the rate of respiration was 2.8 M O2 per min per 107 cells (*R*2 >0.942). Right panel: Lymphocytes (15 x 107 cells/mL) were collected from umbilical cord; the rate of respiration was 8.0 M O2 per min (*R*2 >0.934), or 0.5 M O2 per min per 107 cells. The additions of 5.0 mM NaCN and 50 g/mL glucose oxidase are shown.

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 O2 per min per 107 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 O2 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 O2 per min per 107 cells (Rotig et al., 1990). Hedeskov and Esmann reported a rate of 2.0 + 0.07 nmol O2 per min per 107 for cell concentrations >4 x 107 per mL and higher rates for less concentrated cells (Hedeskov and Esmann, 1966). Pachman reported rate of 1.0 + 0.2 nmol O2 per min per 107 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).

Phosphorescence Oxygen Analyzer as a Measuring Tool for Cellular Bioenergetics 253

Goodisman, J.; Hagrman, D.; Tacka, KA. & Souid, A-K. (2006). Analysis of cytotoxicities of

Gottlieb, E.; Vander Heiden, MG. & Thompson, CB. (2000). Bcl-xL Prevents the Initial

Green, D.R. & Kroemer, G. (2004). The pathophysiology of mitochondrial cell death. *Science*.

Hedeskov, C.J. & Esmann, V. (1966). Respiration and glycolysis of normal human

Jiang, Y.; Jolly, P.E.; Preko, P.; Wang, J-S.; Ellis, W.O.; Phillips, T.D. & Williams, J.H. (2008).

Johnson, WW.; Harris, T.M. & Guengerich, FP. (1996) Kinetics and mechanism of hydrolysis

Jones, E.; Penefsky, HS. & Souid A-K. (2009). Caffeine impairs HL-60 cellular respiration*.* 

Kogelnik, A.M.; Lott, M.T.; Brown, M.D.; Navathe, S.B. & Wallace, D.C. (1997). MITOMAP:

Leist, M. & Wendel, A. (1996). A novel mechanism of murine hepatocyte death inducible by

Lo L-W, Koch CJ, Wilson DF (1996). Calibration of oxygen-dependent quenching of the

Louis H, Moine O L, Peny M-O, Quertinmont E, Fokan D, Goldman M & Devie`re J. (1997).

Margalit, M.; Abu Ghazala, S.; Alper, R.; Elinav, E.; Klein, A.; Doviner, V.; Sherman. Y.;

Marriage, B.J.; Clandinin, M.T.; MacDonald, I.M. & Glenerum D.M. (2004). Cofactor

Meky, F.A.; Hardie, L.J.; Evans, S.W. & Wild, C.P. (2001). Deoxynivalenol-induced

Montesano, R.; Hainaut, P. & Wild, C.P. (1997). Hepatocellular carcinoma: from gene to

public health*. J. Natl. Cancer Inst*. Vol..89, No.24, pp.1844-1851.

*Molecular and Cellular Biology*. Vol.20, No.15. pp.5680–5689.

lymphocytes. *Blood*. Vol.28, No.2. pp. 163-174.

virus disease. *Clin. Dev. Immunol*. (2008:790309).

*Journal of Medical Sciences*. Vol.2, No.2, pp. 61-72.

concanavalin A. *J Hepatol*. Vol.25, No.6, pp.948-959.

*Analytical Biochemistry*. Vol.236, No.1, pp.153-160.

19). pp.257-267.

Vol.305, No.5684. pp. 626-629.

Soc. Vol.118, No.35, pp.8213-8220.

*Acids Res*. Vol.25, No.1, pp. 196-199.

*Hepatology*. Vol.25, No.6, pp.1382-1389.

*Biochemistry*. Vol.313, No.1, pp.137-144.

*Food Chem. Toxicol*. Vol.39, No.8, pp.827-836.

platinum compounds. *Cancer Chemotherapy and Pharmacology*. Vol.57, No.2, (2005 Jul

Decrease in Mitochondrial Membrane Potential and Subsequent Reactive Oxygen Species Production during Tumor Necrosis Factor Alpha-Induced Apoptosis.

Aflatoxin-related immune dysfunction in health and in human immunodeficiency

of aflatoxin B1 exo-8, 9-Epoxide and rearrangement of the dihydrodiol. J Am Chem

an update on the status of the human mitochondrial genome database. *Nucleic* 

phosphorescence of Pd-meso-tetra (4-carboxyphenyl) porphine: A phosphor with general application for measuring oxygen concentration in biological systems.

Production and role of interleukin-10 in concanavalin A–induced hepatitis in mice.

Thalenfeld, B.; Engelhardt, D.; Rabbani, E. & Ilan, Y. (2005). Glucocerbroside treatment ameliorates conA hepatitis by inhibition of NKT lymphocytes. *Am J Physiol Gastrointest Liver Physiol*. Vol.289. No.5, (Epub 2005 Jun 23), pp.G917-25. Marriage, B.J.; Clandinin, M.T.; MacDonald, IM. & Glerum, D.M. (2003). The use of

lymphocytes to screen for oxidative phosphorylation disorders. *Analytical* 

treatment improves ATP synthetic capacity in patients with oxidative phosphorylation disorders. *Molecular Genetics Metabolism*. Vol..81, No.4, pp.263-272.

immunomodulation of human lymphocyte proliferation and cytokine production.
