A spectrophotometric assay for chlorine-containing compounds

ANALYTICAL

BIOCHEMISTRY

196,

262-266

A Spectrophotometric Compounds’

(1991)

Assay for Chlorine-Containing

Jason A. Chesney, Jqhn R. Mahoney, Jr., and John W. Eaton’ Department Received

of Laboratory MedicinelPathology and Dight Laboratories, University of Minnesota, Minneapolis, Minnesota 55455

February

5, 1991

Determinations of hypochlorous acid and chloramine compounds are important in a number of areas. Several techniques are now available for such analyses, but most require unstable reagents and/or multiple steps in the analytical procedure. We have developed a simple, one-step spectrophotometric assay for reactive chlorine-containing compounds involving the oxidation of ascorbic acid by hypochlorous acid or chloramines. There is no interference from other nonhalide oxidants such as hydrogen peroxide or hypothiocyanous acid. Because small amounts of ascorbic acid will not damage biological materials, this method also allows continuous measurements of the generation of chlorine-containing compounds by activated neutrophils. This simple assay permits precise analysis of as little as 1 nmol of HOCl. o 1991 Academic press, Inc.

Reliable techniques for the measurement of reactive chlorine-containing compounds are required for routine monitoring of the adequacy of chlorination of tap water (1). Detection of chlorine-containing compounds may also be important in guarding against, for example, the oxidant damage which occurs to red cells of hemodialyzed patients when dialysis water is inadequately dechlorinated (2). In addition, measurements of chlorine-containing compounds may be important in experimental situations-for example, in estimating the activity of HOCl-producing reactions such as that mediated by the neutrophil enzyme, myeloperoxidase. Currently available techniques for the solution-phase measurement of both hypochlorous acid (HOC1)3 and chlorai This work was supported by Grant RO-1 AI25625 from the National Institutes of Health. x To whom correspondence should be addressed. 3 Abbreviations used: HOCl, hypochlorous acid; DTNB, 5,5’-dithiobis(2-nitrobenzoic acid); HBSS, Hanks’ buffered salt solution; PMA, phorbol myristate acetate; MPO, myeloperoxidase; PBS, phosphatebuffered saline; HOSCN, hypothiocyanous acid; NaSCN, sodium hypothiocyanate; H,O,, hydrogen peroxide; TNB, 5-thio-2-nitrobenzoic acid. 262

mines may work well but often require several discrete analytic steps and are, therefore, unnecessarily cumbersome. In addition, some of these techniques require inherently unstable reagents and are susceptible to interference by nonhalide oxidants. Consequently, we have developed a simple assay based on the facile reaction between HOC1 and ascorbic acid. This reaction yields dehydroascorbate and chloride and is accompanied by almost complete loss of the ultraviolet absorbance of ascorbic acid at 265.5 nm. The substantial molar absorptivity of ascorbic acid at this wavelength (15,000 M-’ cm-‘) (3) makes this assay particularly sensitive. Furthermore, incidental oxidation of ascorbic acid by other agents such as hydrogen peroxide and hypothiocyanous acid is minimal, giving this simple test relatively high specificity for HOC1 and chloramines. MATERIALS

AND

METHODS

Reagents. Potassium phosphate, sodium phosphate, L-ascorbic acid, sodium thiocyanate (NaSCN), sodium chloride, 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB), taurine, potassium cyanide, ethylenediamine tetraacetic acid (EDTA), Hanks’ balanced salt solution (HBSS), Percoll, phorbol 12-myristate 13-acetate (PMA), and hydrogen peroxide (H,O,) were obtained from Sigma Chemical Co. (St. Louis, MO). Ammonium chloride and sodium hypochlorite were obtained from EM Science (Gibbstown, NJ). Sodium borohydride was obtained from Aldrich Chemical Co. (Milwaukee, WI). Sodium chloride was obtained from Fisher Scientific (Fair Lawn, NJ). Hespan (6% solution in isotonic saline) was obtained from the Du Pont Co. (Boston, MA). Prosil-28 was obtained from SCM Chemicals (Gainesville, FL). The eosinophil peroxidase and human neutrophil myeloperoxidase (MPO) were generous gifts from Dr. Arne Slungaard and Dr. Beulah Gray, respectively. Preparation of solutions. Unless otherwise noted, all solutions were prepared in phosphate-buffered saline (PBS) containing EDTA (10 mM sodium phosphate/ 0003-2697/91$3.00

Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

SPECTROPHOTOMETRIC

ASSAY

FOR

150 mM NaClRiO pM EDTA, pH 7.2). EDTA was added in order to chelate reactive transition metals, especially iron and copper, which would otherwise cause spontaneous, metal-catalyzed oxidation of the ascorbic acid (4). In order to further minimize metal-catalyzed ascorbic acid oxidation, we employed water deionized by means of a Millipore mixed bed-carbon ion exchanger water purification system (estimated final resistivity 3 1 MQ). However, we should note that phosphate salts, which were used to prepare the PBS, are well known to be contaminated with trace metals, especially iron (4), necessitating the presence of added EDTA. Stock solutions of HOC1 were prepared by dilution of reagent sodium hypochlorite (730 mM) in PBS, pH 7.2. Stock solutions of H,O, (reagent solution, 8.2 M) were also diluted in PBS, pH 7.2. For preparation of chloramine compounds, in most cases, 9 parts of solutions of 66.67 mM taurine or 66.67 mM ammonium chloride (also in PBS) were admixed with 1 part of 600 mM sodium hypochlorite. Solutions of 6 mM sodium ascorbate were prepared in deionized water. Concentrations of stock solutions of HOCl, monochloramine, and taurine chloramine were determined spectrophotometrically using known molar absorptivities (350 M-’ cm-’ at 292 nm (5), 424 Me1 cm-’ at 242 nm (6), and 398 M-’ cm-’ at 250 nm (7), respectively). In addition, HOCl, monochloramine, taurine chloramine, and HOSCN concentrations were independently determined by coupled oxidation of 5-thio-2-nitrobenzoic acid (TNB) to DTNB as described by Aune and Thomas (8). The TNB was prepared by reducing DTNB with sodium borohydride. In order to prevent subsequent oxidation of the 1 mM TNB solution by metal contaminants and light, 5 mM EDTA was added and the solution container was covered with aluminum foil. Stock H,O, concentrations were determined using a molar absorptivity of 71 M-’ cm-’ at 230 nm (9). Enzymatic and cellular generation of oxidants. Hypothiocyanous acid (HOSCN) was produced by incubating 2.5 PM eosinophil peroxidase with 330 PM H,O, and 450 pM NaSCN for 4 min in 10 mM PBS, pH 7.2, at 25°C. (It has recently been found that NaSCN is the preferred substrate for this enzyme, the reaction yielding 1 mol of HOSCN per mole of H,O, consumed (lo).) In some experiments, MPO activity was followed indirectly by measuring the coupled oxidation of ascorbic acid by HOC1 generated by the reaction between human neutrophil MPO and H,O,. Human neutrophils were prepared as described by Stroncek et al. (11). Care was taken during the preparation to avoid neutrophil activation. Quartz cuvettes used for the neutrophil experiments were siliconized with Prosil-28 to prevent contact activation. Human neutrophils (4.6 X 105) were suspended in 1 ml of HBSS, pH 7.2, containing 60 pM ascorbic acid. Neutrophils were activated with the addition of 50 ng PMA.

CHLORINE-CONTAINING

COMPOUNDS

263

HOC1 assay procedure. Assay of HOC1 via the coupled oxidation of ascorbic acid was performed in the following manner: 10 ~1 of an aqueous stock solution of 6 mM sodium ascorbate (60 pM final concentration) was added to 980 ~1 PBS, pH 5.0 to 8.0, containing 50 pM EDTA, and the initial absorbance was determined spectrophotometrically at 265.5 nm. Varying volumes of the HOC1 standard solutions or unknown samples, usually 10 ~1, were then added to the cuvette and the absorbance read again (30 s after the addition of the HOCl-containing solution). The amount of ascorbic acid oxidized was determined using a molar absorptivity for ascorbic acid of 15,000 M-’ cm-’ (3). In the event that low concentrations of chlorine-containing compounds require the addition of large volumes of sample, the changes in absorbance due to dilution may be corrected mathematically (assuming no spectral activity of the unknown) or by addition of an equal volume of the unknown to a blank cuvette. Chloramine assay procedure. The assay for chloramine solutions was identical to the assay for HOC1 except that the pH of the PBS/EDTA buffer was adjusted to 5.0 (to promote dissociation of chloramine compounds). Even after adjustment of pH to 5.0, the reaction between chloramine-containing solutions and ascorbic acid is substantially slower than that between HOC1 and ascorbic acid. Therefore, in the case of chloramine-containing samples, the absorbance at 265.5 nm was read 5 min after the addition of the sample. Statistics. All data reported here are the means & the standard deviation of three independent determinations. The regression of the lines fit for data shown in Figs. l-3 exceeds 0.99.

RESULTS AND DISCUSSION The oxidation of ascorbic acid to dehydroascorbate is accompanied by almost complete loss of the absorbance peak of ascorbic acid at 265.5 nm and this has been used earlier as a direct measure of ascorbic acid oxidation (12). Possible formation of the semidehydroascorbate radical via one-electron ascorbic acid oxidation is not a problem because this product will quickly disproportionate to form dehydroascorbic and ascorbic acids (13). Addition of increasing amounts of HOC1 to ascorbic acid at pH 7.2 causes a linear, stoichiometric oxidation of ascorbic acid (Fig. 1). This reaction is extremely rapid, being complete within 5 s over a range of pH from 5 to 8. Because of the high molar absorbtivity of ascorbic acid at 265.5 nm, it is possible to detect quite low levels of chlorine-containing compounds. As shown in Fig. 2, micromolar amounts of HOC1 can reliably be measured with this simple technique. Since the oxidation of only 1 nmol of ascorbic acid corresponds to a substantial loss

264

CHESNEY,

0

10

20

30

40

50

60

MAHONEY,

70

HOC1 (nmol) FIG. 1. Stoichiometric oxidation of ascorbic acid by HOCI. Reaction conditions were as described under Materials and Methods. Note that the oxidation of 60 pM ascorbic acid is complete at 60 pM HOC1 (m) and that the addition of excess HOC1 (0) causes no further change in absorbance at 265.5 nm.

in absorbance (0.015 AU), the detection limit for this assay is
AND

EATON

dency of ascorbate to “autoxidize.” This spontaneous oxidation of ascorbic acid is almost entirely due to the presence of contaminating metals, importantly copper and iron (8). In order to eliminate any possible interference by metals, 50 PM EDTA was added to the PBS. In fact, the quality of water to be used for assays of the present type can be determined simply by measuring the spontaneous oxidation of ascorbate in the absence of EDTA (4). As a rule, if the spontaneous rate of oxidation is less than 1 X lo-’ Wmin, addition of EDTA is not necessary. HOC1 is produced in uiuo as one product of the respiratory burst of neutrophils (15). The generation of HOC1 arises from MPO-mediated oxidation of chloride to HOCl, an oxidation dependent on HzO, produced by the respiratory burst. As shown in Fig. 4, MPO activity can be followed by means of the coupled oxidation of ascorbic acid. In the absence of substrate (H,O,), no loss in absorbance occurred, thereby demonstrating that MPO activity was indeed causing ascorbic acid oxidation. Furthermore, as a control, the addition of HOC1 instead of H,O, resulted in the expected loss in absorbance. Activated human neutrophils (106) are capable of producing approximately 2 X 10e7 mol of HOC1 in 2 h (15). As shown in Fig. 5, human neutrophils activated by PMA cause a linear rate of ascorbic acid oxidation. Unactivated neutrophils do not produce HOC1 since the enzyme responsible for the respiratory burst, “NADPH oxidase,” remains dormant (15). Predictably, neutro-

HOC1 (nmol) Reaction Note that causes a 0.015 AU denote f

Oxidation of ascorbic acid by low concentrations of HOCl. conditions were as described under Materials and Methods. as little as 1 nmol HOC1 (volume added to cuvette, 100 ~1) readily detectable change in absorbance (approximately after adjustment for the effect of dilution). Vertical bars 1 SD of three independent determinations.

SPECTROPHOTOMETRIC

ASSAY

FOR

CHLORINE-CONTAINING

265

COMPOUNDS TABLE

1

Oxidation of Ascorbic Acid by HOC1 and Chloramines at pH 7.2 vs pH 5.0 Ascorbic pH Reactants

(60 nmol)

HOC1 Monochloramine Taurine chloramine

-0

10

20

MONOCHLORAMINE

TAURINE

CHLORAMINE

30

40

ADDED

ADDED

hd)

acid

oxidized

(nmol)

7.2

pH 5.0

15 s

5 min

15 6

5 min

59.8 + 0.2 1.1 + 0.7 0.4 + 0.5

59.7 + 0.2 15.2 f 0.9 6.3 + 1.5

60.1 + 0.3 21.4 + 1.9 9.2 * 2.3

60.2 + 0.1 60.9+- 0.3 60.4 2 0.3

HOC1 detected is unaffected by the inclusion of 100 mM H,O,. In the absence of transition metals, H,O, does not react with ascorbic acid and, therefore, provides no interference with the present assay. Even oxidants with reactivities close to those of halides do not interfere with the present assay. For example, the “pseudohypohalous acid,” hypothiocyanous acid (up to 15 PM), causes no oxidation of ascorbic acid. Chlorine-containing compounds are frequently quantitated by means of a reaction with TNB, a reaction which has a variety of drawbacks. In order to obtain TNB one must first reduce 5,5’-dithiobis(2-nitrobenzoic acid) by adding a twofold molar excess of sodium borohydride and incubating for several hours at 37°C with constant shaking (8). Furthermore, the reduced form

hmol)

FIG. 3. Oxidation of ascorbic acid by chloramines. Reaction between monochloramine (A) or taurine chloramine (B) and ascorbic acid at pH 5.0 vs pH 7.2, measured 5 min after combination of the reactants. Reaction conditions were as described under Materials and Methods. Note the slower rate of ascorbic acid oxidation by chloramines at pH 7.2. Vertical bars (barely visible) denote kl SD of six independent determinations.

phils not activated by PMA caused no oxidation of ascorbic acid. The average rate of ascorbic acid oxidation by lo6 activated human neutrophils was found to be approximately 1.3 nmol/min (Fig. 5). This is comparable with the rate of HOC1 production by lo6 activated human neutrophils reported by Weiss (1.6 nmol/min) (15). In the present experiments, oxidation of ascorbic acid by neutrophils started approximately 90 s after PMA activation of the neutrophils-a lag period characteristic of the neutrophil respiratory burst. Importantly, we find little interference by other reduction/oxidation agents. For example, the amount of

CONTROL

10

0

1

2

3

4 TIME

5

6

7

8

9

10

(min)

FIG. 4. Oxidation of ascorbic acid by myeloperoxidase in the presence of HzOz and chloride. Approximately 0.1 U/ml MPO and 60 pM H,O, were reacted in the presence of PBWEDTA buffer, pH 7.2, containing 60 WM ascorbic acid (D). In the absence of added H,O, (0) no reaction occurred whereas direct addition of 60 pM HOC1 caused rapid stoichiometric oxidation of all ascorbic acid (0).

CHESNEY,

MAHONEY,

1 I

I

AND

EATON

We conclude that measurement of solution-phase chlorine-containing compounds via ascorbic acid oxidation is relatively specific and sensitive. This procedure will measure and discriminate between HOC1 and chloramines. The assay is capable of measuring less than 1 nmol of HOC1 and is unaffected by several reducing/oxidizing compounds which might confound analyses by other procedures. We anticipate that this simple, onestep assay procedure will be useful in a variety of circumstances wherein sensitive measurements of the concentrations of reactive chlorine-containing compounds are required. ACKNOWLEDGMENT We thank

Dr. Linus

Ogan

for helpful

discussions.

-0 0

12

3

4

5

6

7

8

9

TIMEbin) FIG. 5. Oxidation of ascorbic acid by activated human neutrophils. Human neutrophils were suspended in HBSS (with Ca*+ and Mg*+), pH 7.2, containing 60 pM ascorbic acid and 50 rig/ml PMA (0). No ascorbic acid oxidation was observed in the absence of PMA (0). Vertical bars denote +l SD of three independent determinations using a simple neutrophil preparation.

(TNB) is unstable and must be kept under nitrogen to prevent oxidation. Finally, since TNB reacts in 2:l stoichiometry with a variety of oxidants, including hypothiocyanous acid, it is a relatively nonspecific method for determining chlorine-containing compounds. A second assay for chlorine-containing compounds measures the formation of periodide spectrophotometritally (16). Chlorine-containing compounds oxidize iodide to iodine which then forms periodide. However, iodide reacts with a number of oxidants including H,O,. Since varying amounts of H,O, are often present in biological systems, catalase must be added to prevent oxidation of the iodide ion. Furthermore, although MPO activity is easily monitored through the coupled oxidation of ascorbic acid, this would obviously be impossible using iodide oxidation because of the interference by the H,O, substrate.

REFERENCES 1. Sawyer, C. N. (1967) Chemistry 366, McGraw-Hill, New York.

for Sanitary

Engineers,

pp. 365

2. Eaton, J. W., Kolpin, C. F., Swofford, H. S., Kjellstrand, and Jacob, H. S. (1973) Science 181,463-464.

C.-M.,

3. Scarpa, M., Stevanato, Chem. 258,6695. 4. Buettner, G. R. (1988)

J. Biol.

R., Viglino,

P., and Rigo, A. (1983)

J. Biochem. Biophys. Methods 16,27-40.

5. Morris, J. S. (1966) J. Phys. Chem. 70,379&3805. 6. Grisham, M. B., Jefferson, M. M., Melton, D. F., and Thomas, E. L. (1984) J. Biol. Chem. 259,10,404-10,413. 7. Weiss, S. J. (1982) J. Clin. Invest. 70, 8. Aune, T. M., and Thomas, E. L. (1977) 214.

59%607. Eur. J. Biochem. 80,209-

9. Beutler, E. (1975) Red Cell Metabolism, p. 89, Gnme New York. 10. Slungaard, A., and Mahoney, J. R. (1991) J. Biol. 4903-4910. 11. Stroncek, (1986) 12. Mahoney,

D. F., Vercellotti,

G. M.,

14. Thomas,

Chem. 266,

P. W., and Jacob,

H. S.

Arteriosclerosis 6, 332-340. J. R., and Graf,

E. (1986)

13. Martell, A. E. (1980) in Autoxidation tems (Simic, M. G., and Karel, M., New York. 15. Weiss, 16. Weiss,

Huh,

& Stratton,

E. L. (1979)

in Food and Biological SysEds.), pp. 89-118, Plenum,

Infect. Zmmun. 25,110-116. Med. 320, 365-376.

S. J. (1989) N. Engl. J. S. J., Klein, R., Slivka,

Invest. 70,598-607.

J. Food Sci. 51,1293-1296.

A., and

Wei,

M.

(1982)

J. Clin.