C H E M I L U M I N E S C E N T D E T E R M I N A T I O N OF 0 2 - AND I o 2
insights into the mechanisms of xenobiotic activation of numerous pharmaceutical and environmental agents. Furthermore, this model of bioactivation may be particularly useful in understanding the observed interaction between inflammation and cancer 1°,31 as well as the mechanisms of toxicity and leukemogenesis in leukocyte-rich organs such as the bone marrow and blood, s,32 Since 90% of the immature granulocytes of the body reside in the bone marrow and contain high levels of myeloperoxidase, this mechanism of xenobiotic activation may be particularly important in this organ. Acknowledgments This work was supported by National Institutesof Health Grants P42 ES04705 and P30 ES01896 and the National Foundation for Cancer Research. David A. Eastmond was supported by an appointment to the Alexander Hollaendcr Distinguished Postdoctoral Program administered by the U.S. Department of Energy and Oak Ridge Associated Universities. Work was pcfforrncd in part under the auspices of the U.S. Department of Energy by the Lawrence Livermorc National Laboratory under Contract W-7405-ENG-48. W e thank Dr. David Ross for valuable contributions.
3t T. W. Kensler,P. A. Egner, K. G. Moore,B. G. Taffe,L. E. Twerdok,and M. A. Trush, Toxicol. Appl. Pharmacol. 90, 337 (1987). 32L. E. Twerdok and M. A. Trash, Chem.-BioL Interact. 65, 261 (1988).
 Determination of Superoxide Radical and Singlet Oxygen Based on Chemiluminescence of Luciferin Analogs By MINORU NAKANO
2-Methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one or 2methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one reacts with 02- or 102 to emit light, probably via the dioxetanone analog. Superoxide dismutase (SOD, a scavenger of 02-) or NaN3 (a quencher of IO2) can be used for differentiation between 02- and IO2-dependent luminescence. The maximum light intensity or integrated light intensity is detected for the assay of 102 or 02- generation in biological systems. METHODS IN ENZYMOLOGY, VOL. 186
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
LEUKOCYTES AND MACROPHAGES
Reagents 2-Methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (CLA) 1 or 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (MCLA), 2 56-60 /~g/ml in doubly distilled water (the solution is stored in 1.0-ml aliquots at -80 ° until needed; CLA or MCLA concentrations are based on e 4 1 0 n m = 8900 M -1 cm -~ and e430nm = 9600 M-I cm-l, respectively) Xanthine oxidase (XO) (grade III buttermilk) (Sigma, St. Louis, MO); activity can be determined by the method described by Roussos 3 Peroxidases, lactoperoxidase (LPO), myeloperoxidase (MPO), chloroperoxidase (CPO), and horseradish peroxidase (HRP); concentrations are based on measured coefficients of 1.14 x 105 M -1 cm -I at 412 nm for LPO, 4 9.2 x 104 M -~ cm -l at 430 nm for MPO, 5 7.53 x 104 M -~ cm -1 at 403 nm for CPO, 6 and 1.02 × 105 M -1 cm -~ at 403 nm for HRP 7 Determination of 0.2-
Estimation of Ability of Human Granulocytes or Monocytes to Generate 02Preparation of Opsonized Zymosan. Zymosan (Sigma), 4.0 mg/ml, is suspended in 20 mM veronal buffer at pH 7.4, boiled for 100 min, and cooled to room temperature. Zymosan is collected after centrifugation for l0 min at 1900 g and then added to pooled human blood serum at a concentration of l0 mg/ml and incubated at 37° for 30 rain. After incubation the zymosan is collected by centrifugation and washed twice with Hanks' balanced salt solution (HBSS) or a modified Hanks' balanced salt solution (mHBSS). The resulting opsonized zymosan (OZ) is suspended to a concentration of 20 mg/ml in HBSS or mHBSS. 2 Preparation of HBSS or mHBSS. HBSS, 0.98% (w/v) (Nissui Pharmaceutical, Tokyo) is dissolved in double-distilled water, and the pH value is adjusted to 7.4 with sodium bicarbonate, mHBSS is prepared by dissolvi S. Inoue, S. Sugiura, M. Kakoi, and T. Goto, Tetrahedron Lett., 1609 (1969). 2 A. Nishida, H. Kimura, M. Nakano, and T. Goto, Clin. Chim. Acta (in press); M. Nakano, this volume . 3 G. G. Roussos, this series, Vol. 12, p. 5. 4 L. P. Hager, this series, Vol 17A, p. 648. 5 j. Schultz and H. W. Shmukler, Biochemistry 3, 1234 (1964). 6 D. R. Morris and L. P. Hager, J. Biol. Chem. 241, 1763 (1966). 7 G. R. Schonbanm and S. Lo, J. Biol. Chem. 24/, 3353 (1972).
CHEMILUMINESCENT DETERMINATION OF 0 2 - AND I o 2
ing HBSS in water as described above and adjusting the pH to 7.4 with 280 mOsm disodium phosphate, instead of sodium bicarbonate, mHBSS is better than HBSS for keeping pH values constant during long incubation experiments. Preparation of Granulocytes and Monocytes. Blood is drawn into a heparinized syringe (20 units/ml) from healthy human volunteers and patients. Leukocytes are isolated by sedimentation in the presence of dextran followed by brief hypotonic lysis of contaminating erythrocytes.8 The resulting leukocytes, which contain 65-91% granulocytes, are suspended (I × 10 7 cells/ml) in HBSS and kept at 0° for no longer than 3 hr prior to use. Human peripheral blood monocytes are prepared according to Kumagai et al. 9 and suspended in HBSS. Preparation of Standard Curve. For the measurement of XO-induced luminescence, the incubation mixture contains different concentrations of XO, 43/~M hypoxanthine, 0.5/xM MCLA (or CLA), and mHBSS to a total volume of 2 ml. After a 3-min preincubation of the mixture without XO and MCLA (or CLA), the reaction is initiated by the simultaneous addition of XO and MCLA (or CLA). During the luminescence measurement, the incubation mixture is agitated by rotation at 37° in the luminescence reader. CLA- or MCLA-dependent luminescence in the XO-hypoxanthine system (corrected for control without the enzyme), in terms of maximum light intensity, is a linear function of XO concentration.l° Thus, the following equation can be obtained: Maximum light intensity corrected for control = factor times XO units (with our chemiluminescence detector). MCLA-dependent luminescence is 4.6 times brighter than CLA-dependent luminescence. The factor will alter depending on the chemiluminescence detector used. Assay o f 02- Generation in Activated Granulocytes or Monocytes. CLA- or MCLA-dependent luminescence is measured with a luminescence reader (Aloka, BLR-101). Reaction mixtures typically contain 04 x 105 granulocytes or monocytes, 1.6 mg OZ, 0.5/zM MCLA or CLA, and HBSS or mHBSS for a total volume of 2.0 ml. In some cases, 0.5 mM NAN3, 0.5/zM SOD, or other substances are added. The volume of each additive (MCLA, NAN3, SOD, or OZ) is 0.1-0.2 ml. All contents, except for OZ and MCLA (or CLA), are preincubated for 3 min, and the reaction is initiated by the simultaneous addition of OZ and MCLA (or CLA). The light emission by CLA or MCLA in the presence of activated granulo8 H. Rosen and S. J. Klebanoff, J. Clin. Invest. 58, 50 (1976). 9 K. Kumagai, K. Itoh, S. Hinuma, and H. Tada, J. Irnmunol. Methods 29, 17 (1979). 10 K. Sugioka, M. Nakano, S. Kurashige, Y. Akuzawa, and T. Goto, FEBS Lett. 197, 27 (1986).
LEUKOCYTES AND MACROPHAGES
cytes or monocytes is inhibited by SOD but not by catalase (20/xg/ml) or 2 mM benzoate. Azide at 0.5 mM does not inhibit the light emission significantly. These results indicate that Oz-, rather than H202, • OH, IO2, or HOC1, is the agent responsible for eliciting the chemiluminescence of CLA or MCLA. TM A suitable standard reaction mixture contains 7 × 104 granulocytes or monocytes suspended in mHBSS containing 0.5/xM MCLA (or CLA) and 1.6 mg OZ (100/zl of serum/mg of zymosan) for a total volume of 2.0 ml. According to the above equation, the maximum rate of 02- production in activated granulocytes or monocytes, corresponding to maximum light intensity, can be expressed as XO units. Twenty normal humans, ages 23-25 years, donated granulocytes which were activated and assayed. Their ability to generate 02- was determined as 217.1 +-- 61.1 (mean -+ S.D.) XO units for the standard MCLA system and 231.5 +-- 69.7 XO units for the standard CLA system, z Caution. MPO (a host enzyme in granulocytes) and hydrogen peroxide can similarly evoke CLA or MCLA luminescence. Treatment of 2.5 × 106 granulocytes/ml with 1 mg OZ results in the release of 1.83 nM MPO (6% of the total MPO) during a 15-min incubation. 11 This amount of MPO could account for only a small fraction (-5%) of the light emission by CLA or MCLA in the presence of OZ-stimulated granulocytes. Thus, it is not necessary to consider the MPO-dependent luminescence, if 4 × 105 cells/2.0 ml are used for the luminescence assay. Of course, granulocytes, which are highly contaminated with colored substance such as hemoglobin, cannot be used for this assay, because of absorbance of the light.
Estimation of 02- Generation in SubceUular Fractions Preparation of Subcellular Fractions. Mitochondrial fractions are prepared from beef heart, n submitochondrial fractions are prepared by an established method, 13 and microsomal fractions are obtained from rat liver. 14 Procedure. The submitochondrial system contains 0.3 mM NADH or 7 mM sodium succinate, 12.5-I00/~g protein (submitochondrial fraction), 4/~M MCLA, 0.25 M sucrose, and 20 mM Tris-HC1 buffer (pH 7.4) for a total volume of 1.0 ml. The volume of each additive (NADH, succinate, or MCLA) is 10/A. The reaction is initiated by the addition of N A D H or succinate. The incubation is carried out at 37° in the chemiluminescence u M. Nakano, K. Sugioka, Y. Ushijima, and T. Goto, Anal. Biochem. 159, 363 (1986). t2 p. V. Blair, this series, Vol. 10, p. 78. 13 H. Low and I. Vallin, Biochim. Biophys. Acta 63, 361 (1963). 14 H. E. May and P. B. McCay, J. Biol. Chem. 243, 2288 (1968).
CHEMILUMINESCENT DETERMINATION OF 0 2 - AND 10 2
== o x I00
0Jg of protein/ml) FIG. 1. Relationship between 02- generation (XO units) and submitochondrJal concentration in the NADH-contaJning system.
reader (Aloka, BLR-102). The 10-min integrated light intensity corrected for control without substrate is calculated 15 and expressed as XO units (Fig. I). The XO units can be obtained using the system containing 43/~M hypoxanthine, XO (50-400 units), 4/zM MCLA, 20 mM Tris-HCl buffer (pH 7.4), and 0.2 M sucrose for a total volume of 1 ml. The chemiluminescence is measured, and 10-min integrated light intensity obtained with 100 units of XO is defined as I unit. The microsomal system contains 1.0 mM NADPH, 10/xM MCLA, microsomes (20-100/zg of protein), and 0.1 M Tris-HCl buffer (pH 7.4) for a total volume of 1.0 ml. The volume of NADPH, succinate, or MCLA is 30/~1. The reaction is initiated by the addition of NADPH after a 3-min preincubation. The incubation is carried out at 37°. The generation of 02in the microsomal systems can be expressed as xanthine oxidase units, identical to the mitochondrial systems. Under the experimental conditions, 02- generation in the microsomal system containing NADPH as substrate is approximately 3.5 times higher than that in the mitochondrial system containing NADH as substrate (Fig. 1). Caution. Submitochondrial and microsomal fractions contain colored materials which absorb the chemiluminescence. Thus, integrated light intensity increases linearly with increasing concentrations of microsomes z5 K. Takayama, M. Nakano, K. Zinner, C. C. C. Vidigal, N. Duran, Y. Shimizu, and G. Cilento, Arch. Biochem. Biophys. 176, 663 (1976).
LEUKOCYTES AND MACROPHAGES
or submitochondria, but only in the range between 20 and 100 /xg of protein in the reaction mixture. Since microsomal and submitochondrial fractions contain cytochrome c-reducing systems, a conventional method for the detection of O2- (cytochrome c method 16) cannot be used. The sensitivity of the chemiluminescence method is about 10 times higher than that of the adrenochrome method, ~6which is widely used for O2- generation in mitochondrial systems. ~7 Determination of 102
Determination of Generation of 102 in Peroxidase-HeO2-Halide Systems Preparation of Standard Curve for 102 Generation in NaOCI-H202 System. The reaction mixture contains 20 mM H202, NaOC1 (10-30/zM), 10/zM MCLA, and 0.1 M acetate buffer (pH or pD 4.5) for a total volume of 1 ml. The reaction is initiated by the rapid injection of 20/zl of NaOC1 20 sec after the addition of 20/zl of MCLA. For experiments in 93.5% D20, apparent pH, measured with a glass electrode, is adjusted to 4.8 to give a pD of 4.5.18 Chemiluminescence is measured in a chemiluminescence reader at 25°. Integrated intensity is calculated 15and plotted against NaOCI concentration. Linearity could be obtained up to 30/~M NaOC1. The integrated light intensity in D20 is about 4 times that obtained in H20. Procedure. The reaction mixture contains 3 nM peroxidase, 20 mM H202,20 mM KBr, 10/zM MCLA, and 0. I M acetate buffer (pH 4.5 or pD 4.5) for a total volume of 1.0 ml. The reaction is initiated by rapid injection of 20/zl of peroxidase using a microsyringe 20 sec after the addition of 20 /zl MCLA. With peroxidases in the range of 1 to 10 nM, MCLA-dependent luminescence, similar to the emission in the NaOC1-H202 system, can be observed in the peroxidase-H202-KBr system and increases linearly with increasing enzyme concentration in both H20 and D20. Chemiluminescence measurements and incubation conditions are essentially the same as those for the preparation of the standard curve. Under these experimental conditions, the replacement of H20 with D20 would enhance the integrated chemiluminescence, i.e., a 4.7 (--.0.6)-fold increase for the LPO system, a 4.3 (-0.3)-fold increase for MPO system, and a 2.8 (+-0.5)-fold increase for CPO. The HRP system does not emit, because no HOBr is produced. On the basis of the generation of 1 mol of IO2 from 1 ~6j. M. McCord and I. Fridovich, J. Biol. Chem. 257, 2713 (1969). t7 j. F. Turrens and A. Boveris, Biochem. J. 191, 421 (1980). is p. Salomaa, L. L. Schaleger, and F. A. Long, J. Am. Chem. Soc. 86, 1 (1964).
CHEMILUMINESCENT DETERMINATION OF 0 2 - AND 102
tool of NaOC1 in the presence of excess of H202 in the H202-NaOC1 systems in D20 and the participation of IO2 generated from the HOBr + H202 reaction in the MCLA-dependent luminescence, the ~O2 concentration generated in peroxidase-H202-KBr systems in D20 can be calculated as 4.6 - 0.9/zM for the LPO system, 12.4 --- 0.6 ~M for the MPO system, and 14.5 -+ 0.6/zM for the CPO system using the standard curve. Caution. Judging from the increase in integrated light intensity in D20 by a factor of more than 4, MCLA-dependent luminescence in the LPO or MPO system containing KBr is considered to be induced only by 102 generated in the system, according to the reaction: H202 + HOBr ~ IO2 + HBr + H20
Most of the emission from MCLA in the CPO-H202-KBr system would be involved in 102 generation, but also partially in another oxidant. MCLA is also oxidized by HOCI, and probably by HOBr, too, without light emission [reaction (I)]. However the HOCI + H202reaction to prodace 102 [reaction (2)] is much faster than reaction (1) and probably also faster than the ~O2 + MCLA reaction [reaction (3)]. Since peroxidaseM C L A + HOCI --~ products HOCI + H202 ~ IO2 + HCI + H20 IO2 + M C L A ~ dioxetanone analog ~ hv
(1) (2) (3)
H202-halide systems do not produce O2-, it is not necessary to consider O2--dependent luminescence in these systems.