Simultaneous determination of 3-hydroxyanthranilic and cinnabarinic acid by high-performance liquid chromatography with photometric or electrochemical detection

ANALYTICAL

BIOCHEMISTRY

200,

273-279

(19%)

Simultaneous Determination of 3-Hydroxyanthranilic and Cinnabarinic Acid by High-Performance Liquid Chromatography with Photometric or Electrochemical Detection Stephan Christen and Roland Stocker1 The Heart Research Institute, Biochemistry New South Wales 2050, Sydney, Australia

Received

July

Group, 145 Missenden Road, Camperdown,

8, 1991

A convenient and rapid method for the simultaneous determination by HPLC of 3-hydroxyanthranilic acid and the dimer derived by its oxidation, cinnabarinic acid, is described. Buffers or biological samples containing these two Trp metabolites were acidified to pH 2.0 and extracted with ethyl acetate with recoveries of 96.6 f 0.5 and 93.4 k 3.7% for 3-hydroxyanthranilic and cinnabarinic acid, respectively. The two compounds were separated on a reversed-phase (C,,) column combined with ion-pair chromatography and detected photometrically or electrochemically. The method was applied successfully to biological systems in which formation of either 3-hydroxyanthranilic or cinnabarinio acid had been described previously. Thus, interferon-y-treated human peripheral blood mononuclear cells formed and released significant amounts of 3-hydroxyanthranilic acid into the culture medium and mouse liver nuclear fraction possessed high “cinnabarinic acid synthase” activity. In contrast, addition of 3hydroxyanthranilic acid to human erythrocytes resulted in only marginal formation of cinnabarinic acid. We conclude that the method described is specific, sensitive, and suitable for the detection of the two Trp metabolites in biological systems. Q 1992 Academic PWB, I~C.

3-Hydroxyanthranilic acid (3HAA)’ is a Trp metabolite formed along the kynurenine pathway initiated by

i To whom correspondence should be addressed. ’ Abbreviations used: BSA, bovine serum albumin; CA, cinnabarinic acid; BHAA, 3-hydroxyanthranilic acid; IDO, indoleamine 2,3dioxygenase; IFN-7, interferon-y; PBMC, peripheral blood mononudodecyltriethylammonium phosphate; TCA, clear cells; Qix, trichloroacetic acid. 0003-2697/92

Copyright All rights

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0 of

by Academic Press, Inc. reproduction in any form reserved. 1992

either Trp 2,3dioxygenase (EC 1.13.11.11) or indoleamine 2,3dioxygenase (IDO, EC 1.13.11.17). 3HAA is the direct precursor of the cyclic dimer cinnabarinic acid (CA), a well-characterized pigment of fungi and insects (1,2) that contains the phenoxazinone structure. The oxidative conversion of 3HAA into CA (Fig. 1) is catalyzed by transition metals (3) and various extracts of biological material possess “CA synthase” activity (4,5). In certain bacteria, such as Streptomyces antibioticus, 3HAA is thought to serve as a precursor for the phenoxazinone-containing actinomycins (6). A biological function of 3HAA as precursor for CA in mammals has not been described. However, 3HAA and CA were reported to be involved in the induction of bladder cancer (7) although more recent reports suggest that urinary excretion of Trp metabolites was a consequence rather than the cause of this disease (8). Interestingly, 3HAA is formed and excreted in relatively large amounts by interferon-y (IFN-y)-primed mononuclear phagocytes (9,lO). We recently reported that some Trp metabolites of the kynurenine pathway, including 3HAA, are powerful antioxidants (11). Since IFN-7 appears to be the principle in uivo inducer of ID0 (12) and a key mediator of inflammation that also primes macrophages for enhanced production of reactive oxygen species (13), we proposed that IDO-mediated formation of 3HAA and other antioxidant active Trp metabolites represents a local defense against oxidative damage. To evaluate the potential contribution of 3HAA to cellular antioxidant defense systems, suitable methods for its analysis are required. As CA is one of the major oxidation products of 3HAA we developed a new assay that allows the simultaneous determination of both Trp metabolites. Several assays for the determination of 3HAA (14) and CA (15) have been described previously; however, 273

274

CHRISTEN COOH

COOH

MOH

-6H+

AND

COOH

K/koA/-ko

3HAA

CA

FIG. 1.

Oxidative

dimerization

of 3HAA

to CA.

those reported for the latter are rather nonspecific or insensitive (e.g., spectrophotometric detection at 450 nm). This study describes selective and sensitive HPLC methods for the rapid and simultaneous determination of CA and 3HAA. Analysis of the two metabolites was achieved using separation on a reversed-phase column combined with ion-pair chromatography, coupled with programmed photometric detection or, for greater sensitivity, electrochemical detection. MATERIALS

AND

METHODS

Chemicals. 3HAA was obtained from Sigma Chemical Co. CA, synthesized as described in (16), was kindly provided by Dr. R. J. W. Truscott (University of Wollongong, New South Wales, Australia). Human recombinant IFN-7 (Biogen Research Corp., Cambridge, MA) was a kind gift from Dr. T. W. Jungi (University of Berne, Switzerland). Sodium acetate (Suprapur) was obtained from Merck, dodecyltriethylammonium phosphate (QiZ, ion-pair reagent) from REGIS Chemical Co. (Morton Grove, IL), RPM1 1640 medium (powder) from Flow Laboratories, guanidine hydrochloride from International Biotechnologies, Inc. (New Haven, CT), and bovine serum albumin (BSA, fraction V) from Calbiothem. LiquiPure (MODULAB) water was used for all aqueous solvents and buffers. The latter were treated with Chelex 100 (Bio-Rad) to remove transition metals. Organic solvents (HPLC grade) were obtained from Mallinckrodt (Mallinckrodt Australia Pty., Ltd.). All other chemicals were of the highest purity available and were obtained from either Merck or Sigma. Cell cultiuation. Human peripheral blood mononuclear cells (PBMC)‘obtained from an adult healthy donor were isolated and cultured (under endotoxin-low conditions) as described by Geczy et al. (17) except that supplementation with additional glutamine and Hepes buffer was omitted. PBMC (5 X lo6 cells/ml) was incubated in 30-ml Teflon vials (Savillex, Minnetonka, MN) in the presence or absence of IFN-7 (1,000 III/ml) in a final volume of 2 ml. Culture supernatants were collected after 72 h of culture and analyzed for 3HAA and CA, as described below. Incubationof erythrocytes with3HAA. Human eryth: rocytes were obtained from fresh venous blood by collection into EDTA-K,-containing vacutainers (Bec-

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ton-Dickinson) followed by three successive washes (600g for 10 min at 15’C) with 100 mM potassium phosphate buffer (pH 7.0) containing 0.14 M NaCl, 5 mM glucose, and 0.5 inM EDTA. In accordance with the method of Tomoda et al. (15), cells were resuspended in the same buffer at 10% (v/v) final concentration and incubated in the dark at 37°C with 3HAA (200 PM). Aliquots of the reaction mixture were taken at different time points and frozen immediately in liquid nitrogen. Erythrocyte lysates, obtained after the samples were thawed, were passed through a Sephadex G-25 column (PD-10, Pharmacia) equilibrated with 75 IIIM sodium phosphate buffer (pH 7.0) in order to separate low-molecular-weight compounds, including CA, from proteins (e.g., hemoglobin). The protein-free fractions collected were analyzed directly for the two Trp metabolites by HPLC. “CA synthase” activity in crude nuclear extract. An extract of a crude nuclear fraction was prepared from mouse liver as described by Subba Rao et al. (5). Briefly, the tissue was homogenized in 2 vol (v/wet w) of 10 mM phosphate-buffered saline (pH 7.0) using a Potter-Elvehjem homogenizer. Following the removal of cell debris and intact cells by centrifugation (5Og for 2 min at 4’C) the supernatant was centrifuged (1OOOg for 10 min at 4°C) and the resulting pellet washed twice with 10 InM phosphate-buffered saline. The pelleted nuclei were then lysed by resuspension in the minimum volume of 75 mM sodium phosphate buffer (pH 7.0) required for subsequent homogenization (10,000 rpm for 3 min at 4°C) in a Polytron homogenizer (PT 3000, Kinematica AG, Switzerland). The resulting lysate was centrifuged again (12,000g for 10 min at 4°C) and the supernatant referred to as crude extract. For measurement of “CA synthase” activity, freshly prepared extract (~0.5 mg protein/ml) was incubated at 37°C in the dark in 75 InM sodium phosphate buffer (pH 7.0) supplemented with 3HAA (100 PM), in the presence or absence of MnSO, (50 PM). Boiled extract (10 min at 100°C) was used as a control to determine the level of non-protein-mediated activity. Aliquots of the reaction mixture were removed at different time points, extracted, and analyzed for 3HAA and CA by HPLC. Samples not analyzed immediately,were acidified with trichloroacetic acid (TCA) (see below), frozen in liquid nitrogen, and kept at -70°C until being processed (within 24 h). 3HAA and CA levels did not change significantly under these storing conditions. “CA synthase” activity was expressed as CA formed per milligram protein and protein was determined by the bicinchoninic acid assay (Sigma BCA-1 kit) using BSA as the standard. Extraction of 3HAA and CA. Buffer, RPM1 1640 medium, cell culture supernatant, or crude extract containing the two Trp metabolites was acidified by the addition of ice-cold TCA to 4% (w/v) final concentra-

HPLC

OF

3-HYDROXYANTHRANILIC

lI

5

CINNABARINIC

275

ACID

(PM-60 pump, 2 LC-4B amperometric detectors) using the column and mobile phase described above. Reductive (for CA) and oxidative (SHAA) potentials were applied between a dual glassy carbon electrode in series and the auxiliary electrode. The current resulting from the electrochemical reaction was measured against a Ag/AgCl reference electrode. Data were recorded and analyzed on a Shimadzu Chromatopac C-R4 A. The cyclic voltammogram of CA (x200 PM) was recorded on a BAS 100 B system using a glassy carbon working electrode and a Ag/AgCl reference electrode.

1

0

AND

L IO

15

Time

(min)

20

25

FIG. 2. Reversed-phase HPLC separation of authentic 3HAA and CA. 3HAA (50 nmol) and CA (5 nmol) were applied onto a deactivated Cis column eluted with 100 mM sodium acetate buffer (pH 5.0)/ CH,CN (W2, v/v) containing Qi2 (0.04%, v/v). A flow rate of 1.0 ml/ min was used and the eluent monitored at 325 and 450 nm for 3HAA and CA, respectively. The arrow indicates the change in wavelength. Peak identification: 1, 3HAA; 2, CA.

tion. The mixture was kept on ice for =5 min and then centrifuged (10,OOOg for 3 min). The pH of the proteinfree supernatant was adjusted by adding small amounts of 5 M NaOH, followed by extraction of 3HAA and CA into 5 vol of ice-cold ethyl acetate. To quantify proteinassociated CA, the TCA precipitate was resolubilized in 6 M guanidine hydrochloride/20 mM potassium dihydrogen phosphate (pH 2.0) prior to extraction with ethyl acetate. After centrifugation (2000g for 10 min at 4°C) the ethyl acetate layer was removed, the solvent evaporated, and the dried material redissolved in HPLC mobile phase. Determination of 3HAA and CA by HPLC. HPLC analyses with photometric detection were performed using a LKB Bromma 2150 pump connected to either a programmable Linear UVIS 204 uv/visible detector or an ABI 1000s diode array detector. 3HAA and CA were separated on a deactivated reversed-phase HPLC column (LC-l&DB, 4.6 mm X 25 cm, 5 pm; Supelco, Bellefonte, PA) eluted with 100 mM sodium acetate buffer (pH 5.0)/acetonitrile (8/2, v/v) containing 0.04% (v/v) Q12 (ion-pair reagent) at 1 ml/min. Separation was carried out at ambient temperature and the eluant monitored at 325 nm (3HAA) and 450 nm (CA). Data were recorded and analyzed using a Nelson Analytical Chromatography System (900 Series Interface and Rev. 5.0 Software). Authentic 3HAA and CA, dissolved in mobile phase, were used as external standards and their peak areas used to quantify the Trp metabolites formed by the biological samples. Solutions of 3HAA were prepared immediately prior to use because of its sensitivity to (aut)oxidation (11). CA solutions were stable for up to 1 month when stored at 4°C. HPLC analyses with electrochemical detection were performed on a BAS 400 liquid chromatography system

RESULTS

AND

DISCUSSION

Determination of 3HAA and CA by HPLC withphotometric and electrochemical detection. Using photomet-

ric detection with programmable wavelength change, 3HAA and CA could be analyzed simultaneously within ~20 min (Fig. 2). The use of a deactivated reversedphase column and the inclusion of the ion-pair agent (dodecyltriethylammonium phosphate) in the mobile phase were required to obtain sufficient separation of CA from 3HAA (not shown). Consistent retention times for CA required extended equilibration with mobile and retention times varied phase (e.g., aovernight) slightly with the use of different batches of columns. The detection limits for 3HAA and CA with photometric detection (Linear UVIS 204) at 325 and 450 nm were 10 and 2 pmol, respectively. More sensitive detection of 3HAA and CA was achieved with electrochemical detection. Figure 3 shows the hydrodynamic voltammograms of 3HAA and CA. The former was detected in the oxidative mode (positive

0 0

0.2

Potential

0.4

0.6

(+V)

FIG. 3. Hydrodynamic voltammograms of 3HAA and CA. In independent experiments 3HAA (40 pmol) or CA (40 pmol) were separated by HPLC as described in the legend to Fig. 2 and monitored with electrochemical detection at different potentials. 3HAA (0) was detected at oxidative and CA (0) at reductive potentials. The inset shows the cyclic voltammogram of CA dissolved in anaerobic 100 mu sodium acetate buffer (pH &O)/CH,CN (812, v/v) containing Q,a (0.04%, v/v). The scan was initiated at +l.O V in the negative direction at 100 mV/s; glassy carbon electrode, 2.0 mm2 surface area.

276

CHRISTEN

AND

potentials) and the latter in the reductive mode (negative potentials), the signals reaching a plateau at = +500 mV and = -200 mV, respectively. At equimolar concentration and identical sensitivity range the maximum response of CA was =50% of that of 3HAA. The cyclic voltammogram of CA, taken in anaerobic HPLC mobile phase, revealed reduction and reoxidation of the phenoxazinone at -166 and -114 mV, respectively (Fig. 3, inset)? The net difference in potential of 52 mV between the cathodic and anodic peak suggests (18) that CA undergoes a reversible one-electron transfer reaction. The maximum anodic current, measured -20 s after the cathodic peak, amounted to =95% of the maximum cathodic current, indicating that the half-life of the putative radical formed during the one-electron reduction of CA is relatively long (~1 min). Reduction potentials similar to that observed for CA and formation of a free radical intermediate have been reported previously for other phenoxazinones (19,20). Using a dual working electrode with the upstream and downstream potentials set at -185 and +500 mV respectively, it was possible to determine CA and 3HAA simultaneously (Fig. 4) with detection limits of 0.2 and 0.1 pmol, respectively, i.e., lo- and loo-fold lower than the corresponding values obtained with the photometric detection. Under these conditions, a peak with a retention time very similar to that of CA was detected on the downstream electrode (peak 2’, Fig. 4B). The compound giving rise to this peak was most likely the one-electron reduction product of CA as the signal was observed only if a reducing potential was applied on the upstream electrode. Increasing the negative potential on the reductive electrode further increased the size of the appearing broad peak (peak U, Fig. 4A), making quantification of CA on this electrode difficult. CA was therefore monitored indirectly by detecting the reduction product (peak 2’) on the oxidative electrode. In summary, the electrochemical HPLC methods for the analysis of 3HAA and CA are suitable for the determination of relatively low concentrations of these two Trp metabolites, whereas photometric detection is the obvious choice when high sensitivity is not required. Both detection systems were applied to different biological systems known to form either 3HAA or CA. Extraction of 3HAA and CA. The efficacy with which CA was extracted into ethyl acetate was strongly

3 These values were obtained after prescanning of CA in acetonitrileiwater (l/l, v/v) containing 50 mM tetraethylammonium perchlorate (19). CA was reduced at a positive potential when the cyclic voltammogram was taken without this electrode pretreatment. However, the net difference in potential between the cathodic and anodic peaks did not change significantly.

STOCKER

0

5

10

Time

15

20

25

30

(min)

FIG. 4. Simultaneous determination of 3HAA and CA by HPLC with electrochemical detection. A mixture of 3HAA (40 pmol) and CA (40 pmol) was separated by HPLC and monitored by electrochemical detection as described under Materials and Methods. Using a dual glassy carbon working electrode in series, CA was detected at -135 mV (A) and 3HAA at +500 mV (B). Peak identification: 1, BHAA, 2, CA, U, unknown.

pH-dependent, the recovery decreasing linearly above pH 3.0 (not shown). A recovery of 71.7 + 10.0% (mean k SD, n = 8) was obtained when CA was extracted from protein-free RPM1 1640 medium previously acidified with TCA and adjusted to pH 2.0 (+-0.1). This pH was chosen for all subsequent extractions, as a lower pH was difficult to control and also affected the HPLC analysis (i.e., the TCA contaminating the ethyl acetate extract caused a reduction in the retention time for CA). Total recoveries of 3HAA and CA extracted from proteincontaining solutions compared to protein-free buffer were identical (Table 1). However, the relative amounts of CA recovered from the TCA-precipitated and redissolved protein increased with increasing protein concentration, e.g., 5.4 + 0.7 and 29.3 f 8.7% (mean f SD, n = 5) for 1 and 10 mg BSA per milliliter buffer, respectively. Filtration of the ethyl acetate-extracted TCA supernatant through O.P-pm nylon filters (Acrodisk, Gelman) reduced the recovery of CA by up to 40%. Therefore, we do not recommend filtering of samples, and data for 3HAA and CA in this and all subsequent experiments were obtained from samples injected onto the HPLC column without filtering.

HPLC

OF

TABLE

3-HYDROXYANTHRANILIC

AND

CINNABARINIC

1

A

Recoveries of 3HAA and CA from 75 mM Phosphate Buffer (pH 7.0)“ Compound*

Location

3HAA

+ BSA (1 mg/ml)

Supernatant Pellet

+ BSA

Supernatant Pellet

CA (1 mg/ml)

277

ACID

Percent 96.5 96.3 0.9 93.4 88.3 5.4

1

recoveryC + t + + + +

0.5 0.8 0.2 3.7 2.3 0.7

a Extractions were carried out at pH 2.0 as described under Materials and Methods. * Concentrations of 3HAA and CA were 100 and 5 PM, respectively. ’ Values represent means + SD of four to six independent determinations.

Formation of CA from 3HAA by biological systems. The suitability of the method described was examined for the extraction and analysis of the two Trp metabolites from either IFN-y-treated PBMC (for 3HAA) or mouse liver nuclear fraction and human erythrocytes (for CA). Endogenous formation of 3HAA by PBMC was determined after 72 h of incubation. 3HAA was detected in the culture supernatants of both untreated (Fig. 5A) and IFN-y-treated cells (Fig. 5B). However, priming of PBMC with IFN-y resulted in approximately a fivefold increase in extracellular 3HAA concentration over that at basal levels (2.18 @M vs 0.43 PM), as has been reported

A

0.4

nA

I

1

Time FIG.

(min)

5. HPLC-EC analysis of 3HAA and CA extracted from PBMC culture supematant. Freshly isolated human PBMC was incubated in the absence (A) or presence (B) of IFN-7 for 72 h and the TCA supematant extracted and analyzed for 3HAA and CA by HPLC as described under Materials and Methods. The chromatograms show the signals obtained on the oxidative electrode after separation of the Trp metabolites. To determine CA in the oxidative mode, a reducing potential of -250 mV was applied on the upstream electrode with the downstream electrode operating at +400 mV. The detection limit for CA was 0.5 pmol under these conditions. The amounts of 3HAA injected in (A) and (B) were 10.8 and 54.4 pmol, respectively. Peak identification: 1,3HAA; X, unknown.

4 X

;.I 0

2 I

5

10

15

20

25

Time (min) FIG. 6. HPLC analysis with photometric detection of 3HAA and CA extracted from biological samples. Mouse liver crude nuclear extract (A) or an erythrocyte suspension (B) was incubated with 3HAA for 2 h at 37OC. Following incubation, the samples were processed and analyzed for 3HAA and CA as described under Materials and Methods. The amounts of Trp metabolites injected in (A) and (B) were 7.4 and 7.3 nmol for 3HAA and 0.72 and 0.05 nmol for CA, respectively. The absorbance ranges chosen for the chromatograms were identical with the exception of the detection of CA in erythrocyte suspension (B), where a 25-times more sensitive range was required. The arrow indicates the change in wavelength. Peak identification: 1, 3HAA; 2, CA, X, unknown.

previously (9). 3HAA was identified by its coelution with a synthetic standard using electrochemical and fluorimetric (9) detection (not shown). CA was not detected in these supernatants, even when the sensitive electrochemical detection was used. Similar results (not shown) were obtained using fewer PBMC or the myelomonocytic cell line THP-1 that has been reported to form 3HAA from Trp (21). Addition of 3HAA to the crude extract of a nuclear fraction resulted in the formation of CA, identified by its coelution with and visible spectrum identical to that of a synthetic standard (Fig. 6A). Approximately 7080% of the 3HAA consumed was converted into CA, of which =60-70% was associated with the TCA supernatant (Fig. 7A) with the remainder present in the TCA precipitate (not shown). Addition of MnSO, (50 PM) to the extract resulted in an increased rate of both 3HAA consumption and CA formation (22) with a yield comparable to that observed in the absence of the transition metal. Incubation of 3HAA with MnSO, alone (i.e., in

278

CHRISTEN

AND

25

J0 5

0

0

30

60

90

120

Time (min)

FIG.

7. CA formation in biological samples. (A) 3HAA (open symbols) was incubated at 37°C in 75 mM NaPO, buffer (pH 7.0) with a crude nuclear extract in the presence (triangles) or absence (circles) of MnSO, (50 PM). Boiled crude extract was used as a control (squares). (B) 3HAA (open symbols) was incubated at 37’C in 100 mM KPO, buffer (pH 7.0) containing 0.14 M NaCl, 5 mM glucose, and 0.5 mM EDTA with (circles) or without (squares) human erythrocytes. Aliquots were taken at different time points and the TCA supematant (A) or protein-free Sephadex G-25 fractions (B) analyzed for CA (solid symbols) as described under Materials and Methods (see Fig. 6 for chromatograms). Data represent typical results obtained in two to three independent sets of experiments. Note the difference in units used to express CA formation in (A) and (B).

the absence of crude extract) also resulted in formation of CA, though only after an initial lag period of -40 min (not shown). However, CA formation induced by M&O, was not dependent on the concentration of the latter, suggesting that only trace amounts of the transition metal are required to accelerate oxidation of 3HAA (23). Boiling the extract almost completely inhibited CA formation, indicating that “CA synthase” activity was protein mediated and not simply due to metal contamination derived from the sample preparation. “CA synthase” activity was associated exclusively with the nuclear fraction as addition of 3HAA to liver homogenate depleted of nuclei, i.e., using the supernatant of the first low-speed centrifugation (lOOOg), did not result in detectable CA formation while 3HAA was consumed very rapidly (not shown). In contrast to the mouse liver nuclear extract and a previous report (15), only small amounts of CA were detected when 3HAA was incubated with human erythrocytes (Fig. 6B). Interestingly, two other compounds

STOCKER

absorbing at 450 nm were detected (peaks X) when the cells were incubated with 3HAA but not when they were incubated with CA, indicating that they are likely derived from 3HAA rather than CA. Within the first 120 min only =2% of the initial 3HAA was converted into CA (~0.7 pmol per milligram protein) and almost half of this rate could be ascribed to nonspecific autoxidation (Fig. 7B). This value is at least 50% lower than that reported earlier (15) and, when expressed on a protein basis, “CA synthase” activity exerted by the erythrocytes was more than four orders of magnitude lower than that observed with crude nuclear extract. No significant amounts of CA were detected in the protein-containing fraction of the erythrocyte lysate after Sephadex filtration. Although the precise mechanism of CA formation remains to be elucidated, our method has demonstrated that incubation of erythrocytes with 3HAA results, together with other products, in only marginal formation of CA. Taken together, these results demonstrate that the extraction and HPLC method described here are useful for the selective and sensitive determination of 3HAA and CA in various biological systems. The simultaneous determination of both Trp metabolites will be advantageous for several investigations in progress in our laboratory, including the evaluation of the contribution of 3HAA to cellular antioxidant defences (11) and the suggested role of the hydrogen peroxide-degrading enzyme catalase in the oxidative formation of CA from 3HAA (24). ACKNOWLEDGMENTS We thank Dr. B. Hibbert and Greg Storrier (School of Chemistry, University of New South Wales, Sydney) for their help with and discussion of the cyclic voltammetry measurements. We also appreciate the kind gifts of CA and IFN-I/ from Drs. R. J. W. Truscott (Wollongong University, New South Wales) and T. W. Jungi (University of Berne, Switzerland), respectively. S. C. is a recipient of an Overseas Postgraduate Research Scholarship from the Department of Employment, Education and Training of Australia, and a Sydney University Postgraduate Research Award.

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OF 3-HYDROXYANTHRANILIC

8. Friedlander, E., and Morrison, A. S. (1981) JNCZ 67,347-351. 9. Werner, E. R., Bitterlich, G., Fuchs, D., Hausen, A., Reibnegger, G., Szabo, G., Dierich, M. P., and Wachter, H. (1987) Life Sci. 41, 273-280. 10. Werner-Felmayer, G., Werner, E. R., Fuchs, D., Hausen, A., Reibnegger, G., and Wachter, H. (1989) Biochim. Biophys. Acta 1012,140-147. 11. Christen, S., Peterhans, E., and Stocker, R. (1990) Proc. N&l. Acad. Sci. USA 87,2506-2510. 12. Bianchi, M., Bertini, R., and Ghezzi, P. (1988) Biochem. Biophys. Res. Commun. 152.237-242. 13. Nathan, C. F., Murray, H. W., Wiebe, M. E., and Rubin, B. Y. (1983) J. Exp. Med. 158,670-689. 14. Elderfield, A. J., Truscott, R. J. W., Gan, I., and Schier, G. M. (1989) J. Chromatogr. 495, 71-80. 15. Tomoda, A., Shirasawa, E., Nagao, S., Minami, M., and Yoneyama, Y. (1984) Biochem. J. 222, 755-760.

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