ASSAY OF FORMATION OR REMOVAL OF OXYGEN RADICALS
nescence method. Glutathione, ascorbate, uric acid, and glucose are also inhibitory of the XOD-induced luminescence at concentrations greater than 4 x 10 -6, 3 × 10-8, 1 × 10-5, and 6 x 1024 M, respectively. However, the concentration of each inhibitor in the reaction mixture corresponds to more than 1 100-fold dilution of the concentration which interferes with the SOD assay. Thus, it is not necessary in the SOD assay to consider the interference by contaminants in the biological samples, provided they are adequately diluted. MCLA in solution is relatively unstable and autoxidizes with light emission at 465 nm. The stock solution of 300/xM MCLA in water (1 ml) should be stored at - 8 0 ° . The solution is then thawed, diluted with water to obtain 15 ~ M MCLA, and should be used as soon as possible thereafter.
 A u t o m a t e d A s s a y o f S u p e r o x i d e D i s m u t a s e in B l o o d
By MARY R. L'ABBI~ and PETER W. F. FlSCtaER The analysis of copper zinc superoxide dismutase (Cu,Zn-SOD) in erythrocytes and extracellular SOD in plasma must be carried out by an indirect method since the substrate (02 ~) is an unstable free radical. The O2-: is generated by xanthine plus xanthine oxidase. The SOD activity is calculated from the rate at which the generated 02 ~ reduces cytochrome c, a reaction which is followed spectrophotometrically. If the activity is high, more of the generated radicals are dismutated and less cytochrome c is reduced, whereas if the activity is low, the opposite is true. 1 The method is adapted to an Abbott VP Super System bichromatic analyzer, and, since it uses small volumes and allows for a relatively large number of samples, it is suitable for use in both clinical laboratories and research laboratories utilizing small animals. 2
Apparatus Abbott VP Super System bichromatic discrete analyzer (Abbott Laboratories Ltd., Diagnostics Division, Mississauga, Ontario, Canada) equipped with a 415/450 nm filter. The Abbott ABA-200 bichromatic analyzer has also been used. 2 1 j. M. M c C o r d and I. Fridovich, J. Biol. Chem. 224, 6049 (1969). 2 M. R. L ' A b b 6 and P. W. F. Fischer, Clin. Biochem. 19, 175 (1986).
METHODS IN ENZYMOLOGY, VOL. 186
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SOD IN BLOOD
Reagents Sodium carbonate buffer (20 mM, pH 10.0) containing 0.1 m M EDTA is prepared by dissolving 2.12 g Na2CO3 in distilled water and bringing the volume to 1 liter (A), and by dissolving 1.68 g NaHCO3 in a similar fashion (B). Solutions A and B are mixed to obtain the buffer. EDTA, 0.372 g (disodium salt, Analar analytical reagent, BDH Chemicals Ltd., Toronto, Ont.), is dissolved in 1 liter of mixed buffer. Ferricytochrome c (50/zM) is prepared by dissolving 123.84 mg (type III from horse heart, Sigma Chemical Co., St. Louis, MO) in sodium carbonate buffer containing EDTA and bringing the volume to 20 ml. Xanthine stock solution (1.0 mM) is prepared by dissolving 15.21 mg of xanthine (purified grade, Fisher Scientific Co., Fairlawn, N J) in sodium carbonate buffer containing EDTA and bringing the volume to 100 ml. Xanthine oxidase (-15 mU/ml) is prepared fresh each day by adding approximately l0/xl of xanthine oxidase (EC 184.108.40.206, grade III, from buttermilk, 29.4 U/ml, Sigma) to 20 ml of sodium carbonate buffer containing EDTA. As the activity of the xanthine oxidase varies, sufficient enzyme is used to produce a rate of cytochrome c reduction resulting in a change in A415of 0.025 per minute in the assay system without any added SOD. The xanthine oxidase is kept in an ice bath during the assay. The reagent mixture is prepared by combining 93 ml of the sodium carbonate buffer, containing EDTA, 2 ml of cytochrome c solution, and 5 ml of xanthine solution. The reagent mixture is stable for several weeks if stored refrigerated. Standards are prepared fresh daily by diluting a stock solution of bovine liver SOD, 15,000 U/ml (Sigma), to a working range of 0.75 to 15 U/ml. One unit is defined as the activity that inhibits the rate of cytochrome c reduction by 50% under the conditions specified for a particular system in a 1 ml reaction volume. 1 SOD extraction solution is prepared by mixing 150 ml of chloroform and 250 ml of ethanol.
Sample Preparation Erythrocytes. Blood is collected in a heparinized microhematocrit capillary tube (75 × 1.5 mm) and stored in an ice bath. The tube is centrifuged in an Autocrit II centrifuge (Fisher) at 13,000 g for 3 min. The tube is cut above the lower sealant and below the buffy coat layer, and the length of this section is recorded. The section of capillary tube containing the erythrocytes is placed in a 2-ml screw-capped tube containing 1.0 ml of cold isotonic saline. The erythrocytes are washed out of the capillary tube by shaking. The piece of tube is discarded and the cells are centrifuged at 15,600 g in a microcentrifuge (Brinkmann Instruments, Rexdale, Ont.) for 2 rain at 4°. The supernatant is discarded, and the cells are lysed by the
ASSAY OF FORMATION OR REMOVAL OF OXYGEN RADICALS
addition of 1.0 ml of cold distilled water. Cold chloroform-ethanol extraction solution (0.4 ml) is added, and the mixture is agitated using a vortex mixer. After centrifugation at 15,600 g for 3 min in the microcentrifuge, the upper aqueous layer is carefully decanted and retained for assay of the SOD activity. The aqueous phase can be stored frozen at - 7 0 ° for several months without any change in activity. The erythrocyte samples, prior to lysing, can be stored frozen for several weeks with no apparent loss of activity. 2 Plasma. Rat plasma SOD activity is determined using the plasma in the microhematocrit tube. The section of the tube containing the plasma iS cut above the buffy layer and is placed in a tube containing 0.4 ml of isotonic saline. The plasma is rinsed out of the capillary tube by shaking. Human plasma SOD activity is measured on undiluted samples collected in conventional ways because the activity is lower than the rat activity and the microhematocrit tube does not yield sufficient sample volume. Plasma samples do not require extraction and can be analyzed directly. Plasma samples can be stored frozen for several months without apparent loss of activity.
Assay Procedure Buffer alone is placed in positions 01 to 04 of the carousel, followed by the standards and the samples. The assay reagent mixture is connected to the instrument reagent line, and the cold xanthine oxidase solution is connected to the auxiliary line. The instrument is programmed as shown in Table I.
Calculations The reading obtained for position 01 is added to the readings for positions 02-04, so that four values are obtained (that for position 01 plus the three sums). These are the changes in absorbance caused by the reduction of cytochrome c in the absence of SOD, or the baseline AA. The mean of the baseline AA is calculated (AAi) and should be approximately 0.050A per 2 min revolution. The change in absorbance for each sample (AAs) is equal to AAj plus the change in absorbance obtained from the instrument for each sample. This latter value is negative since the instrument automatically subtracts AAi. The percentage of the original rate of cytochrome c reduction (Rate %) is calculated for each sample as follows: Rate % = AAdAAi x 100
Rate % is plotted against the activity of the standards on semilogarithmic graph paper (Fig. I). The activity of the samples is read directly from
SOD IN BLOOD
TABLE I VP FOR ANALYSIS IN ERYTHROCYTESOR PLASMA
SETTINGS FOR A B B O T T
Units of measure Sample volume (/,tl)~ Reagent volume (/~l) Filter Reaction type Direction Analysis time (min) Number of revolutions Temperature (°) First revolution read Auxiliary volume (/~l) Assay factor
Absorbance (1.25-2.5) 250.0 415/450 Rate Up 2 3 30 Yes 10.22 Equivalent to filter factor b
Sample volume is dependent on the type of sample being analyzed: 1.25 /~l for rat and human erythrocyte lysate, 2.5/,d for diluted rat and undiluted human plasma. b The instrument determines the filter factor from the filter used.
o Erythrocyte Assay
o~ 602 N 5o c~
4O 3O 2O 10
SOD (Units) FIG. 1. Typical calibration curve of the automated SOD method. Rate % is equivalent to the rate of cytochrome c reduction observed with samples or standards divided by the rate in the absence of SOD multiplied by 100.
ASSAY OF FORMATION OR REMOVAL OF OXYGEN RADICALS
the graph. Alternatively, the activity can be calculated using Eq. (2): Rate % = a + b In(SOD activity)
where, by definition, Rate % is 50 when SOD activity is 1 unit, so that a equals 50. The slope, b, is calculated from the standard curve. The activity of the unknowns is therefore determined with Eq. (3) or (4): Activity = e (Rate%-50)/b df U/ml plasma Activity = e (Rate~-5°)/b df(lOOO/vol. RBC,/xl) U/ml packed cells
for plasma or erythrocytes, respectively, where df is the dilution factor calculated by the instrument. Alternatively, the activity can also be expressed as U/mg hemoglobin for erythrocytes and as U/ml or U/mg of protein for plasma.
Comments The coefficient of variation for SOD in plasma ranged from 3.4 to 3.9% within runs and was 4.5% when a single plasma sample was analyzed over a period of 6 months. The within-run variability for erythrocyte SOD ranged from 3.5 to 5.3%, while variation for analyses performed over 3 days was 5.8%. 2 Recovery of known activities of SOD ranged from 92 to 101% with an overall recovery of 98%. 2 The method is linear for both the erythrocyte assay and the plasma assay from 0.2 to 3.0 units, which corresponds to 0.5 to 10 ng of purified bovine liver SOD (Fig. 1). Typical values for erythrocyte samples from humans, rats, and monkeys are given in Table II.
T A B L E II ERYTHROCYTE S O D IN VARIOUS SPECIES a Activity U / m l p a c k e d cells × 102 Species
Rat Human Monkey
F M M F
11 25 20 20
M e a n -- SD 331 339 107 100
- 31 - 44 -+ 20 +-- 21
U / m g hemoglobin
262-367 255-414 73-149 66-144
100 --_ 9 107 ~ 14 37 - 7 34 ± 7
Reprinted with p e r m i s s i o n f r o m M. R. L ' A b b 6 and P. W. F. Fischer, Clin. Biochem. 19, 175 (1986).
ASSAYS IN W H O L E B A C T E R I A L C E L L S
It is possible to prepare 40 to 60 erythrocyte samples for analysis per day. The automated method allows for the analysis of 25 samples in 6 min in contrast to time-consuming manual methods that require large sample volumes. 3 In the present method, the pH for the assay was increased to 10 from 7.8, 4 thus increasing the sensitivity of the assay for Cu,Zn-SOD 17 times. The reduction of cytochrome c was followed at the more sensitive 415 nm compared to 550 nm used in most assays. 1,3 With these modifications, the sensitivity of the assay was increased 28-fold. Using this method, a standard preparation of bovine liver SOD was found to have an activity of 90,560 U/mg of protein compared to the reported activity of 3200 U/mg. Thus, the units of activity calculated with the present assay are equivalent to 0.035 U reported by Sigma. This does not affect the use of bovine liver SOD as standards since they are used only to calculate the slope. A unit of SOD activity is arbitrarily defined as that activity which will decrease the rate of cytochrome c reduction by 50% under standard assay conditions. 3 L. Floh6 and F. 0tting, this series, Voi. 105, p. 93.
4 C. O. Beauchamp and I. Fridovich, Biochim. Biophys. Acta 317, 50 (1973).
 A s s a y o f S u p e r o x i d e D i s m u t a s e A p p l i c a b l e to W h o l e B a c t e r i a l Cells
By F. S. ARCHIBALD Since 1968 a wide variety of assays for the quantitation of superoxide dismutase (SOD) activity have been proposed and employed. Most are indirect assays requiring both a generator of a known flux of 02- and a chemical detector of the flux. SOD is then quantitated indirectly in a sample by the ability of that sample to reduce the detected O2- flux. Such assays have proved suitable for purified SOD and for most crude, dialyzed broken cell preparations. The present alternatives to this sort of indirect SOD assay, for example, direct viewing of O2- in the far-UV, electron paramagnetic resonance using free radical spin traps such as DMPO (5,5'-dimethyl-l-pyrroline 1-oxide), 19F NMR, stopped-flow spectroscopy, and chemiluminescence, are not practical with the usual broken cell extracts employed and SOD levels found in biological systems. Given the need for a controllable, known flux of 02- and known amounts of an O2- detector in all present assays for SOD in crude cell extracts, it is probably not possible to develop an "intact cell" bacterial METHODS IN ENZYMOLOGY, VOL. 186
Copyright © 1990 by Academic Press, Inc. All fights of reproduction in any form reserved.