Biochi~ic~aBB et Biophysica A~ta ELSEVIER Biochimica et Biophysica Acta 1270 (1995) 173-178

In vitro administration of ergothioneine failed to protect isolated ischaemic and reperfused rabbit heart

Anna Cargnoni h,,, Palmira Bernocchi b, Claudio Ceconi a, Salvatore Curello a, Roberto Ferrari a a Cattedra di Cardiologia, Universith degli Studi di Brescia, Brescia, Italy b Fondazione Clinica del Lavoro, Centro di Fisiopatologia Cardiovascolare 'Salvatore Maugeri', Centro di Gussago, c/o Ospedale O.P. 'Paolo Richiedei', Via Pinidolo, 23, 1-25064 Gussago (BS), Italy Received 22 July 1994; revised 5 December 1994; accepted 7 December 1994

Abstract

Ergothioneine, a natural thiol-containing molecule, has recently been proposed to protect the heart against damage caused by ischaemia and reperfusion. We investigated the possibility that ergothioneine can have a role in maintaining the myocardial thiol/dis- ulfide balance and consequently also a protective effect against ischaemic and reperfusion injury. We used isolated Langendorff-perfused rabbit hearts subjected to 45 rain global and total ischaemia followed by 30 min reperfusion at baseline coronary flow (22 ml/min). Ergothioneine was delivered at 10 -5 M and 10 -4 M 60 rain before ischaemia and during reperfusion. Myocardial damage was determined in terms of mechanical function, creatine kinase (CK) and lactate release, energy phosphate stores and the occurrence of . In our experimental conditions the treatment was unable to prevent myocardial damage. Ergothioneine, independently from the dosage used, failed to: (i) increase recovery of developed pressure upon reperfusion (14.4 + 2.3 mmHg in control hearts vs. 10.3 + 2.9 and 12.5 + 2.3 mmHg in 10 -5 M and 10 -4 M ergothioneine treated hearts, respectively); (ii) decrease the rise in diastolic pressure (44.3 + 4.4 mmHg in control hearts vs. 49.8 + 5.8 and 48.0 -/- 7.7 mmHg in treated hearts); (iii) decrease the release of CK and lactate; (iv) increase the levels of adenosine triphosphate (ATP) and creatine phosphate (CP) in tissue upon reperfusion; (v) maintain ratio between oxidized and reduced forms of adenine nucleotide coenzyme, as index of aerobic metabolism; (vi) prevent the decline of reduced (GSH), or the accumulation of oxidized glutathione (GSSG) as an index of oxidative stress.

Keywords: Isolated heart; Ischaemia; Reperfusion; Ergothioneine

1. Introduction the basis of some experimental observations which pre- sented ergothioneine as protective against H202, several Ergothioneine is a thiol- synthesized by lower radical species and some toxic electrophilic organic eukaryots ( Neurospora crassa and Claviceps purpurea); it molecules [9-14]. It was demonstrated that ergothioneine represents the main component of the thiol pool [1,2]. In prevents oxidation of myocardial myoglobin by H20 2 and mammals, where ergothioneine is present in soluble frac- protects the isolated rat heart from damage caused by tion and reaches high concentrations (mM) in erythrocytes, oxygen-free radicals during reperfusion following is- seminal fluid, and [1,3-6], the source of chaemia. This finding, published by Arduini et al. [14], is ergothioneine is from the dietary intake [1,3-7]. relative to a significant reduction of lactate dehydrogenase The physiological role of this thiolic compound has not (LDH) leakage in an ergothioneine treated group measured been completely explained; nevertheless, there exist sev- in perfusate collected during the reperfusion of isolated rat eral hypotheses [8] regarding it. Recently, a generic antiox- hearts subjected to 15 min of global and total ischaemia. idant activity in mammalian tissue has been proposed for Our study aimed at evaluating the ergothioneine protec- ergothioneine [2]. This metabolic role was suggested on tive effect against myocardial ischaemic and repeffusion injury. Given the presence of a thiol group in its molecule, we * Corresponding author. Fax: + 39 30 2522362. propose ergothioneine as a biological reductant. The hy-

0925-4439/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0925-4439(94)00084-0 174 A. Cargnoni et al. / Biochimica et Biophysica A cta 1270 (1995) 173-178 pothesis that it could participate in the regulation of the checked by an Ellab thermometric probe (model CTD 85) thiol/ exchange underlies the key role that cellu- in the pulmonary artery. They were paced using lar thiols have in maintaining the redox status of cellular suprathreshold rectangular pulses at 0.1 ms duration at a proteins through the reduction of protein and the rate of 180 beat/min. The stimuli were delivered via two quenching of oxidants [15,16]. Thus, the lowering of such platinum electrodes, one attached to the metal inflow a thiol concentration by oxygen-free radicals, as occurs cannula and the other to the ventricular apex. during reperfusion, can lead to serious disturbances in the After a 30 min period of aerobic equilibration (22 cellular regulatory system. ml/min), the hearts were randomly divided into: aerobic Furthermore, the minimal metabolism to which ergoth- hearts (n = 14) which were aerobically perfused for a ioneine in mammalian tissues is subjected after ingestion further period of 135 min; the remaining hearts were made seems significant [5]. Is it possible that ergothioneine ischaemic after 60 min of aerobic perfusion by stopping represents a storage thiolic pool, i.e., a sort of reductant the coronary flow for 45 min. Thereafter, coronary flow equivalents accumulation that the cell could utilize under was re-established for 30 min. oxidative stress conditions? Ergothioneine was delivered at 10 -5 M (group 2, n = 4) In this study, we systematically evaluate the possibility and 10 -4 M (group 3, n = 5) before ischaemia (during the of ergothioneine working as a protective thiol donor in aerobic period of 60 min) and during reperfusion. The isolated Langendorff perfused rabbit hearts subjected to remaining hearts received saline vehicle (group 1, n -- 7). global and total ischaemia followed by reperfusion. We monitored its effect on: (a) energetic metabolism measured 2.2. Left ventricular pressure measurements in terms of anaerobic production of lactate, of high energy phosphates tissue contents (CP, adenosine triphosphate, To obtain an isovolumetrically beating preparation, a ATP; adenosine diphosphate, ADP; and adenosine fluid-filled balloon was inserted into the left ventricular monophosphate, AMP) and of the ratio between oxidized cavity via the atrium. The balloon was connected by a and reduced forms of the adenine nucleotide coenzyme fluid-filled polyethylene catheter to a Hewlett Packard (NAD/NADH); (b) cellular damage measured in terms of transducer (model 1290 A OPT 002). At the beginning of CK leakage; (c) heart mechanical function measured as the experiments, the amount of fluid in the balloon was developed and diastolic pressure; and (d) cellular redox adjusted to obtain a diastolic pressure of < 1.0 mmHg. balance measured as occurrence of oxidative stress. 2.3. Biochemical assays

2. Materials and methods Analysis of coronary effluent Coronary effluent fractions were collected in cooled The reagents used were of analytical grade. The en- vials (0°C) at regular intervals during the entire period of zymes used for the biochemical assays were obtained from reperfusion for CK and lactate determinations. Sigma. Ergothioneine was from Serva Feinbiochemica. CK activity was assayed spectrophotometrically by the method of Oliver [21]; lactate was measured enzymatically using the method of Hohorst et al. [22]. 2.1. Heart perfusion Tissue determinations Male, New Zealand white rabbits (2.0-2.3 kg), main- At the end of the experiments, the hearts were clamped tained on a standard diet, were used. They were treated in with precooled Wollenberger tongs and stored in liquid compliance with the guidelines of the American Physio- nitrogen. For the determination of GSH and GSSG, the logical Society. The animals were stunned by a blow to the tissue ( = 100 mg) was deproteinated with 3 M HC104 and head; the hearts were quickly removed and cooled in the supernatant obtained after centrifugation at 6000 × g perfusion buffer (0°C). After being cleaned of connective for 15 min was neutralized with 2 M KzCO 3. A sample of tissue, the hearts (6.25 + 0.13 g, n = 26) were perfused the neutralized extract was analyzed for total glutathione using the Langendorff technique [17,18], with a modified by the method of Tietze [23]. The reaction mixture (1.0 Krebs-Henseleit buffer containing in mM: NaC1 115; ml) contained: 0.05 M potassium phosphate buffer (pH NaHCO 3 25.0; KC1 4.0; KH2PO 4 0.9; MgSO 4 0.65; CaCI 2 7.4), 1 mM EDTA, 0.1 mM 5,5'-dithiobis-(2 nitrobenzoic 1.7 and D-glucose 11.0 [19,20]. The perfusion solution was acid) (DTNB), 0.15 mM NADPH and an appropriate heated to 37°C, bubbled with 95% 0 2 and 5% CO 2, and volume of sample. After 2 rain of preincubation, the transported at a rate of 22 ml/min to the aortic cannula reaction was initiated by the addition of 1 U of glutathione with a Gilson Minipuls 2 rotary pump. The hearts were reductase and the rate of reduction of the DTNB was jacketed (40-44°C) to provide a constant myocardial tem- continuously monitored at 412 nm; the slope was propor- perature of 37°C independent of coronary flow that was tional to the glutathione concentration over the range of 0 A. Cargnoni et al. / B iochimica et B iophysica A cta 1270 (1995) 173-178 175 to 1 /xM. GSSG was measured as described above after 3. Results the preliminary reaction of GSH with 20 mM N-ethyl- followed by complete removal of unreacted Preliminary control studies confirmed the stability of sulfhydryl reagent with diethylether. the mechanical functions in rabbit hearts when aerobically To determine the content of tissue thiol groups, a perfused by the Langendorff technique for 150 min [20]. portion of the left ventricle was homogenized as described In control hearts, the abolition of coronary flow induced by Sedlack and Lindsay [24] with 20 mM EDTA (pH 4.7), a rapid decline of the developed pressure. The diastolic and filtered on nylon. Samples of homogenates were mixed pressure began to increase progressively after the onset of with 0.2 M Tris-HCl (pH 8.2), 0.01 M DTNB and methanol ischaemia and by 45 min it had increased to 25.9 + 1.5 to a final volume of 10 ml. A reagent blank (without mmHg. Reperfusion resulted in a further increase in the sample) and a sample blank (without DTNB) were pre- diastolic pressure which reached a peak 3 min after reper- pared in a similar manner. The mixture was incubated for fusion. At the end of reperfusion, recovered pressure was 30 rain at room temperature. 21.7% with respect to baseline value (Fig. 1). The absorbance was read at 412 nm, and a molar Administration of ergothioneine at 10 -5 (group 2) and extinction coefficient of 13 600 M-lcm-] was used. 10 -4 M (group 3) had no effect on the mechanical func- The acid-soluble thiol group content (as an expression tion during 135 min of aerobic perfusion and failed to of non-protein sulfhydryl groups = NP-SH) was deter- improve the mechanical function during ischaemia and mined similarly in the supernatant obtained after denatura- reperfusion. After reperfusion, the developed and diastolic tion of the homogenate with ice-cold 50% trichloroacetic pressures were: 14.4 + 2.3 and 44.3 ___ 4.4 mmHg in group acid. Protein SH groups (P-SH) were determined by subtract- GROUP 1-CONTROL ing the acid-soluble from the total SH group content. Aerobia Ischaemia Reperfusion Protein determination was carded out according to Brad- ford [25], using bovine serum albumin as standard. 100 l The extraction of energy phosphates (ATP and CP) and oxidized nicotinamide nucleotide (NAD) was carried out at -180°C in frozen tissue, both ground (mixed with 0.4 N 4O HC104) and homogenized using an Ultra-Turrax. The ho- 2O mogenate was centrifuged at 4000 X g for 10 min at 4°C, 0 and the supernatant, adjusted to pH 6.0-6.5 with 6 N I ..., __ __ ~,,~ ~ , -,5 o ,o ,o 3o 4o 55 ,5 KOH, was used for analysis by high-performance liquid chromatography (Waters 600E multisolvent and a model 990 photodiode array detector). Separations were per- GROUP 2-E 10 -5 M formed on a Supelchem C18 3/.~m RP column (0.46 × 15 loo 1 cm). The mobile phase consisted of a continuous gradient of 60 acetonitrile (2.5-25% v/v) in phosphate buffer from pH 4O 6.0 to pH 5.5. Detection was performed at 205 nm for CP 20 and at 260 nm for ATP [26]. 0 ! , , /IT , , For the determination of reduced nicotinamide nu- -,5 o ,; 3'o ,o 55 .5 ,5 cleotide (NADH), an alkaline extraction carried out with alcoholic potassium hydroxide solution (1 M) was used. GROUP 3-E 10 -4 M pH was brought to 7.8 by slow addition of a 100 1 threthanolamine/phosphate mixture [27]. The supernatant was utilized for the analysis following the same analytical 60 conditions described for ATP, CP and NAD. 40 2O 2.4. Statistical analysis 0

-,5 o ,b 2'o 30 ,o ,5 The data are reported as mean + S.E.M. A one-way analysis of variance was first carried out to test for any minutes differences between all of the groups. If a difference was Fig. 1. Effect of ergothioneine (E) on myocardial function. Mean data established, each group was compared with the control (+ S.E.M.) of the left ventricular (LV) pressures are reported. Ergoth- ioneine was delivered at 10 -5 M (n = 4) and 10 -4 M (n = 5) before group (aerobic control) using Bonferroni's 't-test'. The ischaemia (during the aerobic period of 60 min) and during reperfusion. results were considered to be significant if P < 0.05. The other hearts received saline vehicle (n = 7). 176 A. Cargnoni et al. / Biochimica et Biophysica Acta 1270 (1995) 173-178

® provided by data on the release of CK and lactate shown in < REPERFUSION Figs. 2A and B. During aerobic perfusion, small amounts of CK (from 123.7 + 12.0 initially to 42.4 + 14.7 mU/min per g wet weight at the end of perfusion) and of lactate A 121~- (from 1.89 + 0.16 to 2.10 _ 0.16 /xmol/min per g w.w.) were present in the coronary effluent, independent of the W~ presence of ergothioneine. Fig. 2A shows that the release 0oo. of CK progressively increased during reperfusion and that o'v ~ 600" there were no statistical differences among the three groups. 400~ The release of lactate, reported in Fig. 2B, reached a peak 200- during the first minute of reperfusion and suddenly de- creased until it reached the baseline level, suggesting that O- reperfusion caused a washout of the lactate accumulated 01 3 5 10 20 30 during ischaemia. The release of lactate from group 1, 2 minutes ® and 3 hearts was not significantly different. We investigated the effects of ergothioneine on the 7" < REPERFUSION intracellular thiol pool and its ability to reduce oxidative 6. stress upon reperfusion. The reductant action of ergoth- A q ioneine is thought to depend on an ergothioneine GSH redox couple which, in turn, could be of physiological ¢'[: 4. relevance. Oxidative stress was measured as the myocar- tu-~ 3- dial content of GSH and GSSG, and of protein and non- protein thiol groups [17]. Table 1 shows that ergothioneine does not affect the myocardial content of GSH, NP-SH and P-SH after 60 min of aerobic perfusion. In addition, er- gothioneine failed to prevent the depletion of myocardial thiol groups and the accumulation of GSSG after is- 01 3 5 10 20 30 chaemia and reperfusion. minutes Ergothioneine had no positive effect on energy e~e GROUP 1 - CONTROL metabolism evaluated as the content of high energy phos- o--o GROUP 2- ERGOTHIONEINE 10"5M phates and as the NAD/NADH ratio. As expected [28], ~----o GROUP 3- ERGOTHIONEINE 10"4M for group 1 (control) after 30 min of reperfusion, the levels Fig. 2. Effect of ergothioneine on the reperfusion-induced release of CK of nucleotide in the tissue were low compared to the (A) and lactate (B). The values are given as mean 5: S.E.M. For other details, see the legend to Fig. 1. aerobic values before ischaemia (P<0.001). Ergoth- ioneine treatment (groups 2 and 3) failed to increase the myocardial content of nucleotides. Interestingly, at the end of the reperfusion, the level of ATP was still reduced in all 1; 10.3 +__ 2.9 and 49.8 +5.8 mmHg in group 2; 12.5 ± 2.3 groups while the level of CP had recovered almost to the and 48.0 ___ 7.7 mmHg in group 3 (Fig. 1). No statistically aerobic value (Table 2). This finding is supported by data significant differences were found among the groups. in the literature which show a faster rate of restoration of Further evidence of the lack of ergothioneine efficacy is CP than of ATP resynthesis during the early minutes of

Table 1 Myocardial content of GSH, GSSG, protein- and non-protein-thiol groups Group No. of expts. GSH GSSG NP-SH P-SH TOTAL-SH (nmol/mg prot.) (nmol/mg prot.) (nmol/mg prot.) (nmol/mg prot.) Aerobic control 6 11.61 + 0.42 0.175 ± 0.028 18.8 + 1.6 252.0 -I- 13.4 271.0 -I- 14.8 Aerobic treatment-E 10-SM 4 12.01 5:0.51 0.163 5- 0.018 19.3 + 1.3 265.8 + 10.9 285.9 + 15.1 Aerobic treatment-E 10 -4 M 4 11.35 + 0.80 0.192 ± 0.032 20.1 5- 0.9 249.5 -t- 12.4 290.1 + 18.2 Group 1 7 9.58 5- 0.34 0.270 5- 0.033 a 13.9 + 0.7 b 214.0 + 11.9 b 237.9 + 12.3 Group 2 4 8.31 + 0.31 a 0.250 5:0.043 a 10.6 5:1.2 a 211.2 ___ 16.6 b 221.8 5:18.0 b Group 3 5 8.56 5:1.41 b 0.196 5:0.040 a 11.4 5:2.5 b 173.8 5:18.6 a 185.1 -t- 21.1 a The values pertain to the end of the perfusion period. The data are expressed as mean + S.E.M. E = ergothioneine. a p < 0.01 vs. aerobic control. b p < 0.05 vs. aerobic control. A. Cargnoni et aL / Biochimica et Biophysica Acta 1270 (1995) 173-I 78 177

Table 2 Effects of ergothioneine on myocardial metabolism Group NO. of expts. CP Nucleotide (/zmol/g d.w.) (/.tmol/g d.w.) ATP ADP AMP Ad-Nucl NAD/NADH Aerobic control 6 46.0 _ 3.0 18.5 ± 1.35 3.7 + 0.4 0.98 + 0.25 23,2 + 2.1 23.6 + 1.0 Aerobic treatment-E 10 -5 M 4 43.8 ± 4.1 20.1 ± 2.1 4.1 + 0.6 0.85 + 0.15 25,05 _ 2.85 24.0 ± 1.2 Aerobic treatment-E 10 -4 M 4 49.7 ± 6.2 21.0 _+ 2.5 3.8 + 0.4 0.78 ± 0.31 25.58 +__ 3.21 25.9 _+ 1.6 Group 1 7 34.3 ± 5.6 3.2 + 0.6 " 2.1 + 0.45 b 0.79 + 0.14 6.1 + 1.2 " 6.6 + 1.1 a Group 2 4 27.3 + 6.3 b 2.7 + 0.7 ~ 1.9 ± 0.2 b 1.10 + 0.31 5.7 + 1.2 a 12.1 + 4.8 b Group 3 5 31.3 ± 5.8 3.4 ± 0.6 a 2.6 ± 0.24 1.26 + 0.34 7.3 + 1.2 a 7.0 + 1.5 a

The values pertain to the end of the perfusion period. The data are expressed as mean ___ S.E.M. E = ergothioneine, Ad-Nucl = adenine nucleotides. a p < 0.001 vs. aerobic control. b p < 0.05 vs. aerobic control. reperfusion [29]. Table 2 shows that the NAD/NADH duced release of LDH and CK. This discrepancy can be ratio was unchanged by the administration of ergoth- explained considering several differences in the experi- ioneine. This ratio is an important index of aerobic mental models. First, there is a species difference since we metabolic work. Under aerobic conditions, when the Krebs used rabbits instead of rats. Second, there is a difference in cycle produces NADH and the mitochondrial respiratory the perfusion methodology as we employed a non-recir- chain produces ATP, the NAD/NADH ratio is elevated. culating Langendorff preparation instead of a recirculating On the other hand, ischaemia reduces this ratio [30,31]. perfusion. Third, the duration of ischaemia was substan- tially longer in our model: 45 min instead of 15 rain. The dosage used for ergothioneine was nevertheless the same. 4. Discussion Unfortunately, it is not possible to compare the damage degree induced by these different models as mechanical The reported data indicate that, in our experimental function and amount of CK release were not reported in model, the administration of ergothioneine failed to reduce the paper of Arduini et al. However, their experimental damage caused by reperfusion following ischaemia. In our protocol was designed to test the ergothioneine effect on preliminary plan, we had chosen to administer ergoth- myoglobin rather than to systematically evaluate the ioneine at a concentration of 10 -5 M which is an interme- metabolic interaction of this agent with the myocardial diate dosage between that used by Arduini et al. [14] and thiol pool, and to assess its metabolical implications. the dosage used in our laboratory for other sulfhydryl It is difficult to explain the negative findings about the molecules [17,32]. We also repeated the experiments with effect of ergothioneine. These are not due to an the higher concentration of 10 -4 M, however a further inadequacy of our experimental model as we tested in the increase of dosage was considered to be physiologically same experimental setting the antioxidant capacity of N- not relevant. acetylcysteine and dimercaptopropanol, two sulfhydryl Ergothioneine proved to be unable to improve restora- agents able to significantly improve all the measurements tion in aerobic metabolism and in high energy phosphate considered in this study [17,32]. The inefficacy of ergoth- content as shown by a low value for the NAD/NADH ioneine is most likely due to the poor biological availabil- ratio and reduced ATP and CP levels that persisted even ity of its thiol group which, in aqueous conditions, is after 30 min of reperfusion. Ergothioneine treatment nei- predominantly present as the thione rather than as the thiol ther reduces the membrane damage (given the massive CK form [2] (Fig. 3). release during reperfusion) nor limits the mechanical dete- An increase of the dosage used could possibly over- rioration observed under reperfusion. Finally, ergoth- come the ergothioneine poor thiol availability, since its ioneine did not affect the occurrence of reperfusion oxida- reductant activity is reported in cardiac tissue only when tive stress. It failed to overcome the decline of myocardial

GSH and the accumulation of myocardial GSSG. It did not H H H H participate in the thiol/disulfide metabolism, proving to be C--N C--N~ unable to increase the aerobic myocardial levels of the C__N//~ thiol pool (determined as GSH, NP-SH and P-SH) and the % HCH "<-- H H available cellular reductant equivalents. Consequently, it I +/CH~ I + .CH3 cannot limit the drop in thiol group levels during reperfu- HC--N --ell 3 HC--N .~----CH3 [ _ ~CH~ t _ \CH3 sion. COO COO Our findings are in contrast with data reported by Arduini et al. [14], showing that ergothioneine delivered to Thloll¢ form Thionic form isolated rat hearts significantly reduced the reperfusion-in- Fig. 3. Tautomeric forms of ergothioneine in aqueous solution. 178 A. Cargnoni et al. /Biochimica et Biophysica Acta 1270 (1995) 173-178 used at 2 mM concentration [33]. Further experiments will [11] Dahl, T.A., Midden, W.R. and Hartman, P.E. (1988) Photochem. need to be carried out in order to study the bioavailability Photobiol. 47, 357-362 [12] Hartman, P.E., Hartman, Z. and Citardi, M.J. (1988) Radiat. Res. of the ergothioneine thiol group under physiological and 114, 319-330. pathological conditions. [13] Reglinski, J., Hoey, S., Smith, W.E. and Sturrock, R.D. (1988) J. Biol. Chem. 263, 12360-12366. [14] Arduini, A., Eddy, L. and Hochstein, P. (1990) Arch. Biochem. Acknowledgements Biophys. 281, 41-43. [15] Mannervik, B. and Axelsson, K. (1980) Biochem. J. 190, 125-130. [16] Ziegler, D.M., (1985) Annu. Rev. Biochem. 54, 305-329. This work has been supported by the National Research [17] Ceconi, C., Curello, S., Cargnoni, A., Ferrari, R., Albertini, A. and Council (CNR) target project 'Prevention and control dis- Visioli, O. (1988) J. Mol. Cell. Cardiol. 20, 5-13. ease factors', No. 9100156 pf 41 and by a CNR target [18] Langendorff, O. (1895) Pflugers Arch. Physiol. Mensch. Tiere 61, project on 'Biotechnology and Bioinstrumentation'. We 291-332. [19] Krebs, H.A. and Henseleit, K. (1932) Hoppe-Seyler's Physiol. Chem. thank Miss Roberta Ardesi, Miss Patrizia Martina and 210, 33-66. Miss Michela Palmieri for their expert technical assistance, [20] Ferrari, R., Ceconi, C., Curello, S., Guamieri, C., Caldarera, C.M., and Miss Roberta Bonetti for secretarial help. We wish Albertini, A. and Visioli, O. (1985) J. Mol. Cell. Cardiol. 17, also thank Dr. ssa Antonella Boraso and Dr. Bill Dotson- 937-945. Smith for their assistance with the language. [21] Oliver, T.A. (1955) Biochem. J. 61, 116-122. [22] Hohorst, H.Y., Kreuts, F.M. and Biicher, T. (1959) Biochem. Z. 322, 18-46. [23] Tietze, F. (1969) Anal. Biochem. 27, 502-522. References [24] Sedlack, J. and Lindsay, R.H. (1968) Anal. Biochem. 25, 192-205. [25] Bradford, M.M. (1978) Anal. Biochem. 72, 248-254. [1] Melville, D.B. (1958) Vitam. Horm. 17, 155-204. [26] Ferrari, R., Cargnoni, A., Curello, S., Ceconi, C., Boraso, A. and [2] Hartman, P.E. (1990) in Methods in Enzymology: Ergothioneine as Visioli, O. (1992) J. Cardiovasc. Pharmacol. 20, 694-704. Antioxidant (Packer, L. and Glazer, A.N., eds.), Vol. 186, pp. [27] Klingenberg, M. (1985) in Methods of Enzymatic Analysis: Nicotin- 310-318, Academic Press, London. amide-adenin Dinucleotides and Dinucleotide Phosphates (NAD, [3] Stowell, E.C. (1961) in Ergothioneine (Kharasch, N., ed.), pp. NADP, NADH, NADPH) (Bergmeyer, H.U., ed.), Vol. VII, pp. 462-490, Pergamon Press, New York. 251-284, VCH, Weinheim. [4] Hama, T., Konishi, T., Tamaki, N., Tunemori, F. and Okumura, H. [28] Jennings, R.B. and Steenbergen, C. (1985) Annu. Rev. Physiol. 47, (1972) Vitamins 46, 121-126. 727-749. [5] Mayumi, T., Kawano, H., Sakamoto, Y., Suehisa, E., Kawai, Y. and [29] Taegtmeyer, H., Roberts, A.F.C. and Raine, A.E.G. (1985) J. Am. Hama, T. (1978) Chem. Pharm. Bull. 26, 3772-3778. Coll. Cardiol. 6, 864-870. [6] Kawano, H., Otani, M., Takyama, K., Kawai, Y., Mayumi, T. and [30] Bessho, M., Tajima, T., Hod, S., Satoh, T., Fukuda, K., Kyotani, S., Hama, T. (1982) Chem. Pharm. Bull. 30, 1760-1765. Ohnishi, Y. and Nakamura, Y. (1989) Anal. Biochem. 182, 304-308. [7] Briggs, I. (1972) J. Neurochem. 19, 27-35. [31] Katz, A., Edlund, A. and Sahlin, K. (1987) Acta. Physiol. Scand. [8] Brummel, M.C. (1985) Med. Hypotheses 18, 351-370. 130, 193-200. [9] Kawano, H., Murata, H., Iriguchi, S., Mayumi, T. and Hama, T. [32] Ceconi, C., Curello, S., Cargnoni, A., Boffa, G.M. and Ferrari, R. (1983) Chem. Pharm. Bull. 31, 1682-1687. (1990) Cardioscience 1, 191-198. [10] Hartman, Z. and Hartman, P.E. (1987) Environ. Mol. Mutagen. 10, [33] Arduini, A., Eddy, L. and Hochstein, P. (1990) Free Rad. Biol. Med. 3-15. 9, 511-513.