DETECTIONOF HO" WITHDMSO
peroxide reaction can be found in Ref. 7, where we have also demonstrated that the production of IO2 requires the native enzyme. The emission of ~O2 at 1268 nm can be a reliable diagnostic for the production of IO2 in both photosensitizing and chemiluminescent systems. Its application to biochemical systems has hardly been exploited; however, as the availability of instrumentation increases and the sensitivity of the component detector improves, the detection system is likely to become a standard research instrument.
 D e t e c t i o n a n d Q u a n t i t a t i o n of H y d r o x y l R a d i c a l U s i n g D i m e t h y l S u l f o x i d e as M o l e c u l a r P r o b e B y CHARLES F . BABBS a n d MELISSA GALE STEINER
Numerous methods exist for trapping hydroxyl radicals (HO .) with introduced molecular probes that yield chemically detectable products after reaction with HO ..~-3 Effective probes include 5,5'-dimethylpyrroline N-oxide (DMPO), benzoic acid, methional, 2-keto-4-thiomethylbutanoic acid (KTBA), p-nitrosodimethylaniline, tryptophan, and dimethyl sulfoxide (DMSO). Any of the foregoing compounds is likely to detect hydroxyl radicals in simple test-tube reaction systems that contain minimum concentrations of competing HO. scavengers. The situation becomes more difficult, however, if one wishes to measure hydroxyl radical generation in biological systems containing large amounts of proteins, nucleic acids, purines, sugars, urea, and other compounds that are readily oxidized by HO.. Owing to their high and indiscriminate reactivity, 4 hydroxyl radicals are much more likely to react with various endogenous biological compounds in such systems than with the introduced molecular probe. This difficulty may be summarized conceptually as follows. Supi R. A. Floyd, C. A. Lewis, and P. K. Wong, this series, Vol. 105, p. 231. 2 B. Halliwell and J. M. C. Gutteridge, "Free Radicals in Biology and Medicine," p. 206. Oxford Univ. Press, Oxford, 1987. 3 B. Halliwell, M. Grootveld, and J. M. C. Gutteridge, Methods Biochem. Anal. 33, 59 (1987). 4 N. N. Semenov, in "Some Problems of Chemical Kinetics and Reactivity," Vol. 1. Pergamon, New York, 1958.
METHODS IN ENZYMOLOGY, VOL. 186
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
PRODUCTION, DETECTION, AND CHARACTERIZATION
pose that hydroxyl radicals react in a system containing both substance S and competing substance C as follows: kl HO. + S ~ product P~ k2 H O ' + C -----* product P2
Based on the principle of competition kinetics, 5 the probability that H O . will react with S, rather than C, is given by the fraction k~[HO .]IS] kj[S] = kj[HO.][S] + k2[HO.][C] kj[S] + k2[C]
If we consider S as the introduced molecular probe and C as a lumped parameter representing all the competing scavengers of H O . in a given system, then Eq. (3) describes how the proportion of H O . that is detected is influenced by the effective concentration of competing scavengers present and the rate-lumped rate constant, k:. Values for kz may range from 107 to 1010 M -I s e c - I . 6 Hypothetically, considering [C] for the intracellular environment of a living system as at least 0.I M, taking kl for the desired trapping reaction as 10 9 M -t s e c -1, and estimating k2 for interfering trapping reactions as 10s M -~ sec -~, one finds that the fraction of H O . trapped by 1, 10, and 100 mM concentrations of probe, S, would be 9, 50, and 91%, respectively. Because the exact concentrations of competing H O . scavengers and their rate constants for reaction with H O . are difficult to know in most biological experiments, trapping efficiency is likely to be substantially less than 100%, to a variable and unknown extent. The only means to force efficient scavenging of H O . by probe S is to increase the concentration of S to about 100 mM or greater. 7,8 However, most hydroxyl radical scavengers cannot be tolerated by living systems in such high concentrations. Dimethyl sulfoxide is the exception. It is exceedingly nontoxic and can be tolerated by living systems in up to 1 M concentrations. 9-14 The
s j. W. T. Spinks and R. J. Woods, " A n Introduction to Radication Chemistry," 2nd Ed. Wiley (Interscience), New York, 1976. 6 G. V. Buxton, C. L. Greenstock, W. P. Helman, and A. B. Ross, "Critical Review of Rate Constants for Reactions of Hydrated Electrons, Hydrogen Atoms and Hydroxyl Radicals in Aqueous Solution." Radiation Chemistry Data Center, Radiation Laboratory, University of Notre Dame, Notre Dame, Indiana, 1986. 7 C. F. Babbs and D. W. Griffin, Free Radical Biol. Med. 6, 493 (1989). 8 j. E. Repine, O. W. Pfenninger, D. W. Talmage, E. M. Berger, and D. E. Pettijohn, Proc. Natl. Acad. Sci, U.S.A. 78, 1001 (1981). 9 p. E. Benville, C. E. Smith, and W. E. Shanks, Toxicol. Appl. Pharmacol. 12, 156 (1968). l0 M. M. Mason, in "Dimethyl Sulfoxide" (S. W. Jacob, E. E. Rosen, and D. C. Wood, eds.), p. 113. Dekker, New York, 1971.
DETECTION OF HO" WITH DMSO
median lethal dose (LDs0) of intravenous DMSO in animals ranges from about 4 g/kg body weight (51 mM) to 10 g/kg body weight (128 raM) depending on the species. 1°,~5 This value increases if the drug is diluted with water prior to administration in order to avoid the burning effects caused by the heat of solution of pure DMSO. The compound is rapidly absorbed by all routes, and distributes to all tissue compartments. J6In one remarkable experiment, Benville e t al. 9 studied young salmon and trout totally immersed in DMSO solutions. Fish immersed in 2% (0.26 M) DMSO for 100 days exhibited good appetite and normal weight gain. Chemically, DMSO yields a single, stable, nonradical product, methanesulfinic acid (MSA), on reaction with hydroxyl radical, 17,j8 O
H3C--S--CH3 + HO. ---~ • CH3 + H3C-- - - O H DMSO MSA
in approximately 85% yield ~9 (k = 7 × 109 M -i sec-J). 20 Methanesulfinic acid is a primary product of the trapping reaction, and, indeed, one of the oxygen atoms in the MSA molecule is identical to the oxygen of the original trapped hydroxyl radical. Thus, measurement of MSA accumulation in DMSO pretreated biological systems provides a potential means to capture and count the hydroxyl radicals generated therein. Sulfinic acids (RSOOH) are distinct from sulfonic acids (RSO3H), sulfenic acids (RSOH), sulfones (RISOOR2), and sulfoxides (RISOR2). We have developed a simple colorimetric assay for methanesulfinic acid, 2~ in the presence of high concentrations of unreacted DMSO, which is quite sensitive and easy to perform. The assay is based on the reaction of
H R. R. Maurer, in "The Freezing of Mammalian Embryos" Ciba Foundation Symposium 52, p. 116. Elsevier, Amsterdam, 1977. 12 I. Wilmut and L. E. A. Rowson, Vet. Rec. 92, 686 (1973). ~3S. M. Willadsen, in "The Freezing of Mammalian Embryos" Ciba Foundation Symposium 52, p. 175. Elsevier, Amsterdam, 1977. 14 M. J. Ashwood-Smith, Ann. N.Y. Acad. Sci. 243, 246 (1975). 15 E. R. Smith, Z. Hadidian, and M. M. Mason, Ann. N.Y. Acad. Sci. 141, 96 (1%7). ~6 C. W. Denko, R. M. Goodman, R. Miller, and T. Donovan, Ann. N. Y. Acad. Sci. 141, 77 (1967). 17 C. Lagercrantz and S. Forshult, Acta Chem. Scand. 23, 811 (1%9). ~s S. M. Klein, G. Cohen, and A. I. Cederbaum, Biochemistry 20, 6006 (1981). t9 C. F. Babbs and M. J. Gale, in "Free Radicals: Methodology and Concepts" (C. RiceEvans and B. Halliwell, eds.), p. 91. Richelieu Press, London, 1988. z0 L. M. Dorfman and G. E. Adams, National Standard Reference Data Series 46 (NSRDSNBS46), U.S. National Bureau of Standards, U.S. Government Printing Office, Washington D.C., 1973. 21 C. F. Babbs and M. J. Gale, Anal. Biochem. 163, 67 (1987).
PRODUCTION, DETECTION, AND CHARACTERIZATION
sulfinic acids with diazonium salts, first described by Ritchie et al., 22 namely, CH3SOOH + A r - - N ~ N + ---, H ÷ + A r - - N N - - S O 2 - - C H 3 sulfinic diazonium diazosulfone acid salt (colored. hydrophobic)
The product is a colored diazosulfone, which can be selectively extracted into an organic solvent. Of 22 diazonium salts tested, the one generating the most intense color and having the most desirable practical attributes is Fast Blue BB salt. 2~ The canary yellow reaction product (hmax 425 nm) precipitates from aqueous solutions at high (> 10 mM) concentrations and can be extracted into toluene-butanol (3 : 1) and measured spectrophotometrically. There are limitations to this approach, which are subsequently discussed, but it is offered as an attractive, and low-cost alternative to current methods. The sensitivity of the method is about 10 nmol per sample. Methods
Reagent grade sulfuric acid, n-butanol, acetic acid, toluene, and pyridine are obtained from standard sources. Fast Blue BB salt is obtained from Aldrich Chemical Company (Milwaukee, WI). Disposable Sep-Pak chromatography columns (Cat. 51910) are obtained from Waters Associates (Milford, MA). Standard curves are prepared using authentic methanesulfinic acid, sodium salt, obtained from Fairfield Chemical Company (Blythewood, SC) or authentic benzenesulfinic acid, sodium salt (for color reaction only), obtained from Aldrich. Alternative suppliers of methanesulfinic acid can be found in Chem Sources USA (Directories Publishing Company, Orlando Beach, FL). Sample Preparation
The practical assay of biological material has two phases. The first is the separation of sulfinic acid in the sample from potentially interfering species (sample cleanup). The second is the reaction with the diazonium salt, extraction of the colored product, and direct reading in a spectrophotometer (color reaction). For simple enzyme systems, such as the xanthine oxidase system, run in the presence of 5% DMSO, no cleanup 22 C. D. Ritchie, J. D. Saltiel, and E. S. Lewis, J. Am. Chem. Soc. 83, 4601 (1961).
DETECTION OF HO" WITH DMSO
procedure is required. 7 The assay of DMSO-pretreated plant or animal material requires either extraction of the sulfinic acid from interfering materials (method A, to follow) or removal of the interfering materials from the sample (method B). Plant material can be assayed after quickfreezing with liquid nitrogen, grinding with a mortar and pestle, aqueous extraction of the resulting powder, and, if necessary, concentration of the aqueous extract by lyophilization. Bacterial suspensions and animal tissues can be prepared for assay by homogenation and centrifugation. Most of the sulfinic acid remains in the supernatant. Some procedure to remove protein usually improves results. We have found that acidification with HC1 and neutralization with NaOH prior to high-speed centrifugation are effective for this purpose. Trichloroacetic acid precipitation of protein cannot be used, because the trichloroacetate anion produces prodigious interference. A c i d - B u t a n o l Extraction o f Sulfinic A c i d (Cleanup A) Principle. Sulfinic acid (pKa - 2 ) 23,24 is extracted from tissue homogenate into n-butanol at pH 0, followed by backextraction of sulfinate ions into aqueous acetate buffer at pH 4 to 5. Potentially interfering species, including proteins and amines, are precipitated or remain in the original aqueous phase. This step should be completed quickly so as to minimize the dwell time of the sulfinic acid below pH I, since dismutation to the sulfonic acid occurs gradually under these extremely acidic conditions.2L 26 Procedure. Tissue is homogenized in a chilled Teflon-glass homogenizer in 2 volumes of distilled water. A 2.0-ml aliquot of the aqueous sample, expected to contain 10 to 300/~M sulfinate, is placed in a test tube, and 0.2 ml of I0 N sulfuric acid is added with vortexing. The tubes are centrifuged to remove coagulated protein and the supernatants decanted into fresh tubes. Optionally, the supernatants may be extracted once or twice at this stage with 2.0 ml of toluene-butanol (3 : l) to remove interfering detergentlike substances, and the organic layers discarded, after which methanesulfinic acid remains in the water phase. Four milliliters of butanol, previously saturated with 1 M sulfuric acid, is added and mixed thoroughly for 60 see on a vortex mixer. The upper butanol phase
23c. I. M. Stirling,J. Int. J. Sulfur Chem. Part B 6, 277 (1971). 24j. G. Baldirlus, "Treatise on AnalyticalChemistry,Part 2 Vol. 15: AnalyticalChemistry of Inorganicand Organic Compounds"(I. M. Kolthoffand P. J. Elving,eds.), Sect. B-2, Wiley, New York, 1976. 2~j. L. Kice and K. W. Bowers, J. Am. Chem. Soc. 84, 605 (1962). 26E. E. Gilbert, in "InterscienceMonographson Chemistry"(F. A. Cotten, ed.), p. 228. Wiley, New York, 1969.
PRODUCTION, DETECTION, AND CHARACTERIZATION
is removed with a Pasteur pipet to a second, clean test tube containing 2.0 ml of 0.5 M sodium acetate buffer, pH 5, and mixed vigorously. After centrifugation at 500 g for 3 rain, the color reaction can then be run on the sulfinic acid extracted into the aqueous acetate phase. The time taken for this extraction procedure should be minimized and kept similar for all samples and standards.
Removal of Interference with Sep-Pak Columns (Cleanup B) Principle. The ingenious Sep-Pak approach was conceived by Dr. Jean Blair Smith. Many potentially interfering species are anionic detergents, such as bile salts or free fatty acids, which form saltlike complexes with Fast Blue BB dye (hmax 395 rim) that are extractable into toluene-butanol. 19 Most interfering anions are much less hydrophilic than methanesulfinic acid itself. To remove them, preliminary extraction of the sample with toluene-butanol (3 : 1) at neutral pH is helpful, but not always sufficient. Further removal of detergentlike interference can be obtained by adding Fast Blue BB dye to the sample and applying the mixture to a lipophilic (C~8 Sep-Pak) column. When the column is subsequently eluted with water, the complexes of dye and lipophilic anions remain on the column, while sulfinic acid is readily eluted and collected, lit would appear from this and other observations that reaction (5) is quite reversible in aqueous media.] In this way detergentlike anions that are specifically capable of complexing with Fast Blue BB dye to form lipophilic salts are sequestered on the column. The color reaction with additional Fast Blue BB dye and toluene-butanol extraction is then run on the effluent fraction containing the sulfinic acid. Procedure. Tissue samples are homogenized in 3 volumes of distilled water. To precipitate protein the pH is lowered to 1 with concentrated HCI, the sample is allowed to stand 10 min, and the pH is returned to 7.4 with NaOH. Denatured proteins are removed by centrifugation. Twomilliliter aliquots of the supernatant are extracted twice with 2 ml of toluene-butanol (3 : 1) and the organic phases discarded. Then 100/zl of 30 mM Fast Blue BB salt is added to the aqueous sample with mixing, and the tubes are allowed to stand for 10 min in the dark. A 1-ml sample is applied to a Sep-Pak (C~8) column that has been preeluted with 2 ml of methanol and 2 ml of water. The methane sulfinate anion is then eluted from the column with distilled water. The first 1.3 ml of effluent is discarded, and the next 1.5 ml is collected for assay by the color reaction. Color Reaction. A consistent 1 to 2 ml volume of sufficiently clean aqueous sample is transferred to a test tube, the pH is adjusted to 2.5 by the addition of up to 0.3 ml of 0.1 or 1 N HC1, and the color reaction is
DETECTION OF HO" WITHDMSO
begun by the addition of 0.1 ml of 30 mM Fast Blue BB salt (freshly prepared and kept in the dark). The stock solution is stable for at least 8 hr at room temperature. If necessary, stock solutions of BB dye can be extracted with chloroform to remove photooxidized products that can cause high blanks. Ten minutes is allowed for product development at room temperature in the dark. Then 1.5 ml of toluene-butanol (3 : 1) is added and mixed thoroughly with the aqueous phase for 120 sec on a vortex mixer. After low-speed centrifugation to separate the phases, the lower phase, containing unreacted diazonium salt, is removed by aspiration and discarded. The toluene-butanol phase is washed with 2 ml of butanol-saturated water for 30 sec to remove remaining unreacted diazonium salt. The tubes are centrifuged at 500 g for 3 min, and the upper phase, containing the diazosulfones, is transferred to a cuvette. One tenth milliliter of pyridine-glacial acetic acid (95 : 5) is added to stabilize the color, which otherwise fades gradually at acid pH. The bright yellow color is reasonably stable after pyridine addition, fading about 6% per day at room temperature. The absorbance as a function of wavelength from 340 to 520 nm is recorded on a strip chart recorder, using a blank prepared beginning with 2 ml of distilled water carried through the same procedure. In most experiments, the sulfinic acid content may then be calculated from the absorbance at 425 nm, with reference to a standard curve.
Derivative Spectroscopy In some applications double-derivative analysis of absorbance spectra z7-29may be useful to eliminate false-positive interference that does not exhibit a peak (or nadir) at 425 nm, and which can be characterized as a linear function of wavelength from 400 to 450 nm. The spectral region from 400 to 450 nm in the neighborhood of the absorption peak of authentic methanesulfinic acid is digitized, taking at least 5 points, and represented as a second-order polynomial, centered at 425 nm, calculated by a least-squares method. 3°,3j The parabolic curve fit for the regression function A = a0 + al Ah + a2(Ah)2
of absorbance (A) as a function of wavelength (Ah = h - 425) is accom27 T. C. O'Haver and G. L. Green, A n a l Chem. 48, 312 (1976). 28 T. C. O'Haver, Clin. Chem. 25, 1548 (1979). z9 F. P. Corongiu and A. Milia, Chem.-Biol. Interact. 44, 289 (1983). 30 N. R. Draper and H. Smith, "Applied Regression Analysis," p. 17. Wiley, New York, 1966. 31 HP-65 STAT PAC 1, Hewlett-Packard Company, 1974.
PRODUCTION, DETECTION, AND CHARACTERIZATION
plished in our laboratory with the aid of a " C " language computer program, which calculates the least-squares regression coefficients a0, a~, and a2, for n > 1 data points, y = f ( x ) , according to the following expressions: A-B
az = [n E x/z - (E xi)Z][n E x 4 - (E x2)2] - [n E x] - ( ~ x i ) ( ~ X2)] 2 (7) where A = [n ~ x/2 - (E xi)2][n E x2yi - (~i, x~)(E Yi)] B = [n ~, x] - ( ~ xi)(~, x/2)][n ~ xiYi - (~, xi)( E Yi)] al =
[n E xiYi - (E xi)(E Yi)] - a2[n "2 x3i - ( ~ xi)C2 x2)] n ~ x7 - ( ~ x,) 2
ao = - (E Yi - a2 ~ x 2 - al E xi) n
(8) (9) (10) (11)
The second derivative of the spectrum of standard methanesulfinic acid is nearly constant from 400 to 450 nm and is directly proportional to sulfinic acid concentration. F r o m the values of the second derivative of the sample absorbance spectrum at 425 nm, obtained by differentiation of the fitted, least-squares polynomial, the methanesulfinic acid content of the sample is computed as 2aE(sample) Vtot' Y = 2a2(standard) --V amtstd
where A is absorbance, 2a2 the second derivative of the parabolic curve fit at 425 rim, amtstd the amount of standard methanesulfinate assayed (nmols), V the volume assayed, and Vtot the total volume of the sample. This measure of sulfinic acid content is insensitive to interference that is a constant or linear function of wavelength in the neighborhood of 425 nm. Remarks D i a z o n i u m C o u p l i n g R e a c t i o n . Considering the general reactivity of diazonium salts, 32 the diazonium coupling reaction for sulfinic acid is surprisingly free o f interference, a~ Although many substances are known to couple with diazonium salts to produce liposoluble products, 33,34 most
32R. F. Muraca, in "Treatise on Analytical Chemistry" (P. J. E[ving, ed.), Part 2, Sect. B-2, Vol. 15, p. 251-234. Wiley, New York, 1976. 33K. Venkataraman, "The Chemistry of Synthetic Dyes," Vol. I. Academic Press, New York, 1952. 34K. Venkataraman, "The Chemistry of Synthetic Dyes," Vol. 4. Academic Press, New York and London, 1971.
DETECTIONOF HO" WITH DMSO
of these coupling reactions are classically de.scribed at neutral or slightly alkaline pH. 32 The sulfinic acid coupling reaction is unusual in that it proceeds well at acid pH. The probable mechanism for the coupling reaction, by analogy with that described by Hauser and Breslow, 35 is attack of the unbonded electrons of the sulfur by the Ar--N~--N + ion, followed by transfer of the oxygen-associated electron of the sulfinate anion to the sulfur atom. The availability of the unbonded electron of sulfinate anions at pH 2.5 may explain the specificity of the reaction for sulfinic acids as opposed to sulfoxides and sulfonic acids. Other well-known diazonium coupling reactions 32,36-3s with phenols, indoles, and amines (abundant in free amino acids and proteins) proceed best at slightly alkaline pH. By allowing the diazonium coupling to take place in acidic solution, the interference from these species is greatly reduced) 1 Nevertheless, false-positive results can be produced by anionic detergents and sulfite. Causes of False-Positive Results. Anionic detergents in the sample, such as bile salts, generate the most troublesome false-positive interference with the color reaction, by forming lipophilic salts with the diazonium ( A r - - N = N +) cation. This type of artifactual signal typically has an absorbance peak at 395 nm (that of the native Fast Blue BB dye), clearly to the left of the 425 nm peak for authentic sulfinic acid. A good example of this phenomenon can be observed experimentally by simply mixing a detergent such as sodium lauryl sulfate with Fast Blue BB and extracting with toluene-butanol (3 : 1). A small admixture of detergent interference with true sulfinic acid signal will cause a shift of the absorbance peak to the left. Removal of detergent anions by procedures such as solvent extraction or ion-exchange chromatography may be necessary in detergentladen systems such as rat liver. In the presence of detergents the signalto-noise ratio is a strong function of the amount of BB dye added for the color reaction, since the final dye concentration required for half-maximal color reaction of true methanesulfinic acid is about 1 raM, whereas the dye concentration for half-maximal detergent interference is often 10 m M or greater. Exploration of the inherent trade-offs may be worthwhile in some applications. One interesting variant of detergent interference is provided by sodium sulfite. Sulfite reacts rapidly with Fast Blue BB under acidic conditions to produce what we surmise is the sulfonic acid derivative, Ar--N~---N--SOzH. This species, in turn, probably forms salts with unreacted dye (Ar--N~---N ÷) to form a strongly absorbing complex, extract35 C. R. Hauser and D. S. Breslow, J. Am. Chem. Soc. 63, 418 (1941). 36 R. Wistar and P. D. Bartlett, J. Am. Chem. Soc. 63, 413 (1941). 37 j. B. Conant and W. D. Peterson, J. Am. Chem. Soc. 52, 1220 (1930). 3s K. H. Beyer and J. T. Skinner, J. Pharmaeol. Exp. Ther. 68, 419 (1940).
PRODUCTION, DETECTION, AND CHARACTERIZATION
able into toluene-butanol. This effect may even be exploitable to measure sulfite in some applications. It can be minimized by cleanup procedures A and B and by treatment of the final organic phase with dry, beaded ionexchange resins. J9 Another possible source of false-positive results that we have found is artifactual DMSO oxidation during the assay procedure itself, as opposed to biological DMSO oxidation by H O . during the experiment preceding the assay. Suitable combinations of heat, iron, oxygen, reductants such as ascorbic acid or thiols, and/or prolonged incubation or storage times can induce oxidation of DMSO by nonbiological mechanisms to give a product spectrally indistinguishable from methanesulfinic acid in the colorimetric assay. 39 Even though such autoxidation yields less than 0.1 mM methanesulfinic acid from 1 M DMSO, the effect can be of the same order as biologically induced DMSO oxidation. This experimental difficulty is easily detected, however, by a so-called late DMSO addition control experiment, in which DMSO is added to the system after biological oxygen radicals could not be produced (e.g., after depletion of substrate, after denaturation of enzymes) but prior to the assay procedure. If oxidation of DMSO during the assay is found, reasonable steps can be taken to minimize the effect, including use of fresh, reagent grade DMSO, elimination of iron from buffers and reagents, 4°,41 or use of shorter, colder storage conditions for samples prior to assay. Causes of False-Negative Results. The possibility that sulfinic acids may be degraded in biological samples before they can be measured is worthy of consideration and is easily tested by incubation of standard methane sulfinate with the biological system under study for various periods of time. Recovery of added standard from the system is an important validation step, since some sulfinate may become bound to tissue proteins and resist extraction. As previously mentioned, acid-catalyzed dismutation of methanesulfinic acid to methanesulfonic acid, which is not detected by the assay, 21 does occur slowly, so that the duration of acidification of samples during any cleanup procedure should be minimized and kept consistent for standards and samples. Certainly, samples should never be stored in an acidified state. If necessary, calibration can be done using the method of standard addition. In some systems with high concentrations of competing scavengers of HO., even 1 M DMSO may trap a fraction of nascent H O . substantially less than unity.7 This effect can be detected by studying results for a range 39 D. W. Griffin, Masters thesis, Purdue University, Lafayette, Indiana, 1988. 4o j. M. C. Gutteridge, FEBS Lett. 214, 362 (1987). 41 G. R. Buettner, J. Biochem. Biophys. Methods 16, 27 (1988).
DETECTIONOF HO" WITH DMSO
of DMSO concentrations added to the biological system under study. If necessary, Scatchard analysis 4z may be performed to account for the fraction of HO. radicals that react with endogenous compounds rather than DMSO. 7 Summary Generation of clearly harmful amounts of hydroxyl radicals in biological systems can be studied using DMSO as a molecular probe. DMSO is oxidized by HO. to form the stable, nonradical compound methanesulfinic acid, which is not normally found in living systems and which can be easily extracted from tissue and measured spectrophotometrically. The present method provides a simple, inexpensive assay for methanesulfinic acid in biological materials. As little as 10 nmol of sulfinate can be detected, and interference from diverse biological compounds is minimal. Additionally, there is no interference from a large excess of dimethyl sulfoxide, which is necessary if the assay is to be applied directly to tissues pretreated with DMSO. When straightforward cleanup procedures are utilized, there is minimal interference from glutathione or sulfate, and potentially troublesome interference from detergentlike substances can usually be minimized. Owing to its relative specificity for sulfinic acids at acid pH, the diazonium coupling reaction can thus be exploited to provide an efficient and inexpensive means of detecting methanesulfinic acid in DMSO-pretreated biological materials. The results provide a direct chemical means for measuring cumulative HO. generation. Acknowledgments The technical assistance of Ms. Joanne Cusumano and Ms. Meloney Cregor is gratefully acknowledged. Supported by Grants HL-36712 and HL-35996 from the National Heart, Lung, and Blood Institute, by Grant CA-38144 from the National Cancer Institute, U.S. Public Health Service, Bethesda, Maryland, and by a Focused Giving Grant from Johnson & Johnson.
42 G. Scatchard, Ann. N.Y. Acad. Sci., 51 (1949).