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Ergothioneine Distribution in Bovine and Porcine Ocular Tissues
Comp. Biochem. Physiol. Vol. 117C, No. 1, pp. 117–120, 1997 Copyright 1997 Elsevier Science Inc.
ISSN 0742-8413/97/$17.00 PII S0742-8413(96)00223-X
Ergothioneine Distribution in Bovine and Porcine Ocular Tissues Thomas K. Shires,1 Marvin C. Brummel,2,3 Jose` S. Pulido,4 and Lewis D. Stegink3 1
Departments of Pharmacology, 2 Pediatrics, and 3 Biochemistry, College of Medicine, The University of Iowa, Iowa City, IA 52242, U.S.A., and 4 Eye Institute, Medical College of Wisconsin, Milwaukee, WI 53226, U.S.A. ABSTRACT. Ergothioneine (ERT), is a low molecular weight, sulfur-containing antioxidant occurring in up to millimolar amounts in mammalian tissues. Using an improved HPLC assay, ERT levels have been measured and compared in bovine and porcine eyes and erythrocytes. The rank order of ERT levels in bovine ocular tissue was lens . retina 5 cornea . pigmented retinal epithelium (RPE) . aqueous humor (AQ) . vitreous humor (VIT) . sclera. In porcine ocular tissue, the rank order was retina . AQ . VIT . RPE . cornea . lens . sclera. ERT levels in bovine lens were about 250 3 . that in porcine lens. Porcine erythrocyte levels were 5.5 3 . bovine levels. Species differences were also observed in the retina, VIT and AQ where porcine levels were 2 to 10-fold greater than bovine levels. ERT in bovine lens and cornea was 35 and 14 times greater than the corresponding level of reduced glutathione (GSH). Porcine lens had 45 times more GSH than ERT. Values for ERT and GSH in other tissues from both species were of the same order of magnitude. These results are consistent with a role for ERT in prevention of oxidative damage to the eye. comp biochem physiol 117C;1:117–120, 1997. 1997 Elsevier Science Inc. KEY WORDS. Ergothioneine, glutathione, antioxidant, free radical
INTRODUCTION Ergothioneine (ERT) is α-N,N,N-trimethyl-2-thio-histidine (Fig. 1A). It is synthesized exclusively in true fungi and some bacteria, yet occurs widely in human and animal tissues (3,5,11). ERT has escaped more extensive study for two reasons. First, as a quaternary amine, ERT does not possess the primary alpha amino group required for reactivity in most assays (ninhydrin, OPA, FDNB) for amino acids. Second, at physiologic pH, ERT exists mostly in its thione form (Fig. 1A) and as a result, does not readily react well with common sulfhydryl reagents such as DTNB (5,11). ERT can react with oxidants, transferring either one or two electrons; single electron donation results in the formation of the ERT free radical (Fig. 1B), which in turn may be available for either reversible reduction back to ERT, or reaction with another ERT free radical (or thiyl radical) formed from another available sulphydryl forming homo or mixed disulfides of ERT (Fig 1C). Alternatively two moles of ERT can reversibly donate two electrons and two protons in a classical thiol/disulfide oxidation/reduction reaction. The ERT free radical generated by the reaction of ERT with hydroxyl, azide, and trichloromethyl peroxyl radicals can form a one electron redox couple with vitamin C (2). ERT Address reprint requests to: Thomas K. Shires, Department of Pharmacology, The University of Iowa, 2312 Bowen Science Building, Iowa City, IA 52242-1109. Tel. 319-335-7959; Fax 319-335-8930. Received 1 August 1996; accepted 4 November 1996.
also reacts with the peroxynitrite, hypochlorite, and triiodide anion preventing the effect of these oxidants on certain amino acids including tyrosine, tryptophan, methionine, cystathionine, and homocystine (8). The Eo′ for the ERT thiol/disulfide two electron half reaction is 20.06 volts compared with 20.20 to 20.30 volts for other natural thiols such as reduced glutathione (GSH) (9). ERT that has been oxidized in the process of reducing some oxidant (RXN 1) can, therefore, be reduced back to its thiol (thione) form by reaction with GSH (RXN 2). The oxidized glutathione (GSSG) formed can be reduced back to its thiol form by the action of glutathione reductase in the presence of NADPH (RXN 3). RXN 1: 2ERT-SH 1 X ox → ERT-S-S-ERT 1 Xred RXN 2: ERT-S-S-ERT 1 2 G-SH ↔ 2 ERT-SH 1 G-S-S-G RXN 3: G-S-S-G 1 NADPH 1 H1 ↔ NAD1 1 G-SH These one and two electron couples with ERT may give ERT a possible integrating role in the antioxidant pathways of vitamin C and glutathione. A portion of tissue ERT may be bound to protein (11), and previous reports of tissue ERT levels have been based
T. K. Shires et al.
118
FIG. 1. Oxidation-Reduction forms of ERT: (A) thione; (B)
free radical; (C) disulfide.
on extraction followed by assay using analytical techniques that may not have included protein-bound ERT, or that would not have discriminated between ERT and compounds of closely related chemical structures. This led us to develop a method for assaying total tissue ERT using reverse-phase HPLC. Little information on ERT levels in ocular tissue is available, aside from its reported occurrence in calf (4) and human lenses (15). In this report, using improved methods for isolation and analysis of ERT, ERT was detected in major tissues of the eye at levels similar to, and in some cases exceeding, those of glutathione. MATERIALS AND METHODS Freshly enucleated eyes from six Charolais steers or nine Poland China pigs (eight gelds and one sow) were immediately chilled in crushed ice and dissected within 45–60 min. A packed red blood cell sample was prepared from each animal’s blood. Resected corneas, lens, retinas and retinal pigmented epithelium (RPE), and packed red blood cells were weighed, the volumes of vitreous and aqueous humor from each eye were measured, and packed red cell preparations from blood samples were made, immediately prior to freezing each sample in liquid nitrogen until analyzed. Individual
frozen samples were suspended in sufficient cold ethanolic extraction medium (EXM) to give 1 : 10 (sample : EXM, v : v). EXM consisted of 10 mM dithiothreitol (Sigma Chemical Co., St Louis, MO), 100 µM betaine (Sigma Chemical Co.) and 100 µM 1-mercapto-2-methyl imidazole (MMI; Sigma Chemical Co.) in 70% ethanol. MMI served both as an internal standard and, along with betaine, as an agent to disrupt noncovalent ERT-protein interactions. Ethanolic extraction and DTT facilitate disulfide reduction, deproteinization and inactivation of factors that destroy ERT in aqueous homogenates of some samples (e.g., mushrooms and human breast milk). Sample suspensions were individually homogenized with 30 strokes in a Potter-Elvehejm homogenizer. Sufficient ethanolic sodium dodecylsulfate (SDS) was then added to obtain 0.2%, and the suspension gently mixed. Insoluble material was removed by centrifugation in a Sorval HB4 rotor (2000 3 g, 10 min) and the supernatant frozen and lyophilized. Samples were stored at 270°C under high purity nitrogen. Authentic ERT, formerly available from Sigma Chemical Co., was either a gift from Donald Melville or isolated from mushrooms by the method of Melville (11). For analysis by HPLC, each lyophilite was suspended in 0.5 ml (pH 7.3) of aqueous 50 µM thiourea and 100 µM choline (to limit thiourylene and quaternary ammonium ion binding to tissue protein) and filtered through a Centricon-10 ultrafiltration unit (Amicon Corp., Beverly, MA). Separation was carried out on two Econosphere C18 columns (Alltech Associates, Deerfield, IL; each column 250 mm 3 4.6 mm, five µ particle size connected in tandem). Several other ODS-type columns tested did not retard ERT satisfactorily. The isocratic elution buffer was 50 mM sodium phosphate, pH 7.3, pumped at ambient temperature and a flow rate of 1 ml/min using a Beckman 110B HPLC solvent delivery module and a 160 Absorbance Detector equipped with 254 nm filters. The concentration of glutathione in each sample was determined by chromatography of a 50 µL aliquot from the resuspended lyophilite of each sample on a Beckman 6300 amino acid analyzer system using glucosaminic acid (Sigma Chemical Co.) as an internal standard. The resolution of ERT from other compounds present in ethanolic extracts of ocular tissues using the HPLC method described is shown in Fig. 2. The method was linear through 10 mM; the practical lower limit of ERT detection was 0.5 µM. The identity of the peak listed as ERT was confirmed by co-chromatography with authentic ERT and by treatment of the sample with trace quantities of I2, which selectively removes 2-thioimidazoles from the sample, followed by rechromatography of the treated sample using the same HPLC system (Fig. 2). RESULTS AND DISCUSSION Recoveries of internal standards averaged $97% for both bovine and porcine aqueous humor and vitreous humor
Ergothioneine in Ocular Tissues
FIG. 2. HPLC Separation of ERT from Retinal Extracts: (A)
preparation of authentic ERT; (B) extract of bovine retina; and (C) extract of porcine vitreous. The arrow indicates the eleution position of ERT. The insert A shows the effect of elemental iodine on the ERT peak (upper tracing: unoxidized, lower tracing: iodine treated).
samples; $95% for pigmented retinal epithelium and retina; $90% for lens; and $87% for cornea. ERT was detected in all ocular tissues examined (Table 1). Bovine lens ERT was present at concentrations about 17- to 25-fold larger than those in cornea and retina, respectively. ERT concentrations in porcine lens were considerably lower than concentrations found in bovine lens. The range of bovine lens value is large (SD about 60% of mean); an explanation for this variation is not apparent. Porcine retina and pigmented retinal epithelium concentrations of ERT were several-fold greater than values in bovine retina and pigmented retinal epithelium and notably greater than
119
lens concentrations. Although the ERT content of pig lens was markedly lower than that of the cow lens, the ERT content in pig erythrocytes was about 53 greater than the ERT concentration in bovine erythrocytes. Erythrocyte ERT concentrations, which are thought to reflect the amount of ERT in the diet at the time of hematopoiesis (5), suggest that overall dietary intake of ERT was approximately 53 larger in pigs than in cows. Concentrations of reduced glutathione (G-SH) were of the same order of magnitude as ERT concentrations in most of the bovine ocular tissues examined (Table 1). Exceptions were bovine lens, where ERT occurred at concentrations about 353 greater than G-SH, and the cornea where ERT concentrations were about 143 greater than those of GSH. In the pig, G-SH and ERT concentrations were similar in the vitreous humor and the aqueous humor, cornea, perhaps the retina and pigmented retinal epithelia, but in the porcine lens G-SH was about 453 greater than ERT concentrations. To assess whether higher ERT levels in the tissues of one species were balanced by lower G-SH concentrations in the same tissues of other species, and vice versa, values for ERT and G-SH of each tissue were summed (5RSH) and the cow/pig ratios of these sums compared (Table 1). While the ratios were relatively constant for four of seven ocular tissues, the large variation (8.45, for instance for the lens) discounted the notion of balance. The role of oxidative mechanisms and antioxidants in the development and control of various pathologies, including those of the eye, is widely recognized. In the lens, a high level of G-SH appears important for maintenance of normal transparency (13). There is an inverse relationship between extent of cataract formation and ERT concentration in the human lens (11). ERT inhibits lipid peroxidation (1,6), hydroxyl radical formation (2,12), singlet oxygen generation (6,14), and the rate of peroxide formation (5). Reactivity of ERT with hydroxy- and α-halogenated peroxy radicals measured by pulse radiolysis is superior to G-SH (7). Given the antioxidant properties of ERT, its presence in ocular tissues at concentrations approximating those of G-SH argues for consideration of this thiourylene as a relevant biological antioxidant mechanism. The cause of the marked species-specific differences in ocular tissue content of ERT is not clear. Gender and age may be discounted, since most animals were male and all about the same age. However, the diet at the time of hematopoiesis is known to affect ERT concentration in the erythrocyte. Porcine concentrations of RBC ERT were greater than bovine levels, suggesting pigs were better provided with dietary ERT than the cattle. Reliable information on the feeding regimen of commercially slaughtered animals is difficult to estimate. Thus, comparison of the level of ERT uptake with tissue levels was not possible. Shukla et al. (15) have reported human lens levels of ERT of about 5 µmoles/ g tissue, a level intermediate between the values we ob-
T. K. Shires et al.
120
TABLE 1. Distribution of ergothioneine and glutathione in tissues of the eye*
Bovine Tissue Aqueous humor** Vitreous humor† Retina† PRE† Lens† Cornea† RBC‡
ERT 1.2 0.12 2.96 0.49 7.79 4.46 0.04
(0.50) (0.01) (0.20) (0.39) (4.72) (1.34) (0.08)
Porcine
GSH
ERT 1 GSH
0.17 (0.05) 0.09 (0.01) 2.41 (0.05) 0.77 (0.05) 2.33 (0.05) 0.31 (0.05) NA
1.37 (0.55) 0.21 (0.01) 5.37 (0.25) 1.26 (0.39) 10.1 (5.99) 4.77 (1.43) NA
ERT 0.76 1.00 8.69 2.97 0.20 0.93 0.22
(0.26) (0.45) (1.57) (1.10) (0.11) (0.48) (0.18)
Ratio
GSH
ERT 1 GSH
Cow/Pig
0.56 (0.18) 0.08 (0.02) 5.28 (0.91) 5.14 (0.47) 9.30 (0.49) 0.56 (0.14) NA
1.32 (1.04) 1.08 (0.19) 14.0 (0.38) 8.11 (0.16) 9.50 (8.45) 1.49 (3.20) NA
1.04 0.19 0.38 0.16 1.06 3.20 NA
*Values shown are means from six steers or nine pigs with standard deviations shown in parentheses. ** µmoles/mL. † µmoles/gm tissue. ‡ µmoles/gm RBC. NA 5 not analyzed. PRE 5 pigmented retinal epithelium.
served for cows and pigs. Until more is known about the mechanism of ERT uptake and distribution to tissues, the significance of the differences in tissue levels will remain obscure. Supported in part by the Logan Research Fund, Research to Prevent Blindness; a grant from the Mead Johnson Nutritional Group; and by the Children’s Miracle Network Telethon. The assistance of Heather Jackson and Gwen Knapp is gratefully acknowledged, as is the help of Mel and Neva Herschberger of the Kalona Packing Plant, Kalona, IA.
References 1. Akanmu, D.; Cecchini, R.; Aruoma, O.I.; Halliwell, B. The antioxicant action of ergothioneine. Arch. Biochem. Biophys. 288:10–16;1991. 2. Asmus, K.D.; Bensasson, R.V.; Bernier, J.L.; Houssin, R.; Land, E.J. One-electron oxidation of ergothioneine and analogues investigated by pulse radiolysis: Redox reaction involving ergothioneine and vitamin C. Biochem. J. 315:625–629; 1996. 3. Brummel, M.C. In search of a physiological function for Lergothioneine—II. Med. Hypotheses 30:39–48;1989. 4. Calam, D.H. Some new compounds in the lens. Exp. Eye Res. 1:436–442;1962. 5. Hartman, P.E. Ergothioneine as an antioxidant. Meth. Enzymol. 259:310–318;1990. 6. Hartman, P.E.; Hartman, Z.; Ault, K.T. Scavenging of singlet molecular oxygen by imidazole compounds: High and sustained activities of carboxyl terminal histidine dipeptides and exceptional activity of imidazole-4-acetic acid. Photochem. Photobiol. 51:59–66;1990.
7. Jovanovic, S.V.; Simic, M.G. Free radical chemistry of ergothioneine, a potential radioprotector and antimutagen. In: Nygaard, O.F.; Upton, A.C. (eds). Anticarcinogenesis and Radiation Protection. New York: Plenum Press; 1991:229 – 235. 8. Kooy, N.W.; Nelson, S.E.; Brummel, M.C.; Stegink, L.D. Ergothioneine inhibits peroxynitrite-mediated oxidation and nitration reactions. Abstracts. Eighth Biennial Meeting of International Society for Free Radical Research, Barcelona, Spain, 1996:92. 9. Lewin, S. Vitamin C: Its Molecular Biology and Medical Potential. London: Academic Press; 1976. 10. Melville, D.B.; Lubsche, R. A method for the determination of ergothioneine in blood. J. Biol. Chem. 200:275;1953. 11. Melville, D.B. Ergothioneine. Vitamin Horm. 17:155–204; 1959. 12. Motohashi, N.; Mori, I. Thiol-induced hydroxyl radical formation and scavenger effect of thiocarbamides on hydroxyl radicals. J. Inorg. Biochem. 26:205–212;1986. 13. Rathbun, W.B. Glutathione in ocular tissues. In Dolphin, D.; Poulson, R.; Avramovic, O. (eds). Glutathione: Chemical, Biochemical and Medical Aspects, Part B. New York: Wiley & Sons; 1989:467–510. 14. Rougee, M.; Bensasson, R.V.; Land, E.J.; Pariente, R. Deactivation of singlet molecular oxygen by thiols and related compounds, possible protectors against skin photosensitivity. Photochem. Photobiol. 47:485–489;1988. 15. Shukla, Y.; Kulshrestha, O.P.; Khuteta, K.P. Ergothioneine content in normal and senile human cataractous lenses. Indian J. Med. Res. 73:472–473;1981.
ISSN 0742-8413/97/$17.00 PII S0742-8413(96)00223-X
Ergothioneine Distribution in Bovine and Porcine Ocular Tissues Thomas K. Shires,1 Marvin C. Brummel,2,3 Jose` S. Pulido,4 and Lewis D. Stegink3 1
Departments of Pharmacology, 2 Pediatrics, and 3 Biochemistry, College of Medicine, The University of Iowa, Iowa City, IA 52242, U.S.A., and 4 Eye Institute, Medical College of Wisconsin, Milwaukee, WI 53226, U.S.A. ABSTRACT. Ergothioneine (ERT), is a low molecular weight, sulfur-containing antioxidant occurring in up to millimolar amounts in mammalian tissues. Using an improved HPLC assay, ERT levels have been measured and compared in bovine and porcine eyes and erythrocytes. The rank order of ERT levels in bovine ocular tissue was lens . retina 5 cornea . pigmented retinal epithelium (RPE) . aqueous humor (AQ) . vitreous humor (VIT) . sclera. In porcine ocular tissue, the rank order was retina . AQ . VIT . RPE . cornea . lens . sclera. ERT levels in bovine lens were about 250 3 . that in porcine lens. Porcine erythrocyte levels were 5.5 3 . bovine levels. Species differences were also observed in the retina, VIT and AQ where porcine levels were 2 to 10-fold greater than bovine levels. ERT in bovine lens and cornea was 35 and 14 times greater than the corresponding level of reduced glutathione (GSH). Porcine lens had 45 times more GSH than ERT. Values for ERT and GSH in other tissues from both species were of the same order of magnitude. These results are consistent with a role for ERT in prevention of oxidative damage to the eye. comp biochem physiol 117C;1:117–120, 1997. 1997 Elsevier Science Inc. KEY WORDS. Ergothioneine, glutathione, antioxidant, free radical
INTRODUCTION Ergothioneine (ERT) is α-N,N,N-trimethyl-2-thio-histidine (Fig. 1A). It is synthesized exclusively in true fungi and some bacteria, yet occurs widely in human and animal tissues (3,5,11). ERT has escaped more extensive study for two reasons. First, as a quaternary amine, ERT does not possess the primary alpha amino group required for reactivity in most assays (ninhydrin, OPA, FDNB) for amino acids. Second, at physiologic pH, ERT exists mostly in its thione form (Fig. 1A) and as a result, does not readily react well with common sulfhydryl reagents such as DTNB (5,11). ERT can react with oxidants, transferring either one or two electrons; single electron donation results in the formation of the ERT free radical (Fig. 1B), which in turn may be available for either reversible reduction back to ERT, or reaction with another ERT free radical (or thiyl radical) formed from another available sulphydryl forming homo or mixed disulfides of ERT (Fig 1C). Alternatively two moles of ERT can reversibly donate two electrons and two protons in a classical thiol/disulfide oxidation/reduction reaction. The ERT free radical generated by the reaction of ERT with hydroxyl, azide, and trichloromethyl peroxyl radicals can form a one electron redox couple with vitamin C (2). ERT Address reprint requests to: Thomas K. Shires, Department of Pharmacology, The University of Iowa, 2312 Bowen Science Building, Iowa City, IA 52242-1109. Tel. 319-335-7959; Fax 319-335-8930. Received 1 August 1996; accepted 4 November 1996.
also reacts with the peroxynitrite, hypochlorite, and triiodide anion preventing the effect of these oxidants on certain amino acids including tyrosine, tryptophan, methionine, cystathionine, and homocystine (8). The Eo′ for the ERT thiol/disulfide two electron half reaction is 20.06 volts compared with 20.20 to 20.30 volts for other natural thiols such as reduced glutathione (GSH) (9). ERT that has been oxidized in the process of reducing some oxidant (RXN 1) can, therefore, be reduced back to its thiol (thione) form by reaction with GSH (RXN 2). The oxidized glutathione (GSSG) formed can be reduced back to its thiol form by the action of glutathione reductase in the presence of NADPH (RXN 3). RXN 1: 2ERT-SH 1 X ox → ERT-S-S-ERT 1 Xred RXN 2: ERT-S-S-ERT 1 2 G-SH ↔ 2 ERT-SH 1 G-S-S-G RXN 3: G-S-S-G 1 NADPH 1 H1 ↔ NAD1 1 G-SH These one and two electron couples with ERT may give ERT a possible integrating role in the antioxidant pathways of vitamin C and glutathione. A portion of tissue ERT may be bound to protein (11), and previous reports of tissue ERT levels have been based
T. K. Shires et al.
118
FIG. 1. Oxidation-Reduction forms of ERT: (A) thione; (B)
free radical; (C) disulfide.
on extraction followed by assay using analytical techniques that may not have included protein-bound ERT, or that would not have discriminated between ERT and compounds of closely related chemical structures. This led us to develop a method for assaying total tissue ERT using reverse-phase HPLC. Little information on ERT levels in ocular tissue is available, aside from its reported occurrence in calf (4) and human lenses (15). In this report, using improved methods for isolation and analysis of ERT, ERT was detected in major tissues of the eye at levels similar to, and in some cases exceeding, those of glutathione. MATERIALS AND METHODS Freshly enucleated eyes from six Charolais steers or nine Poland China pigs (eight gelds and one sow) were immediately chilled in crushed ice and dissected within 45–60 min. A packed red blood cell sample was prepared from each animal’s blood. Resected corneas, lens, retinas and retinal pigmented epithelium (RPE), and packed red blood cells were weighed, the volumes of vitreous and aqueous humor from each eye were measured, and packed red cell preparations from blood samples were made, immediately prior to freezing each sample in liquid nitrogen until analyzed. Individual
frozen samples were suspended in sufficient cold ethanolic extraction medium (EXM) to give 1 : 10 (sample : EXM, v : v). EXM consisted of 10 mM dithiothreitol (Sigma Chemical Co., St Louis, MO), 100 µM betaine (Sigma Chemical Co.) and 100 µM 1-mercapto-2-methyl imidazole (MMI; Sigma Chemical Co.) in 70% ethanol. MMI served both as an internal standard and, along with betaine, as an agent to disrupt noncovalent ERT-protein interactions. Ethanolic extraction and DTT facilitate disulfide reduction, deproteinization and inactivation of factors that destroy ERT in aqueous homogenates of some samples (e.g., mushrooms and human breast milk). Sample suspensions were individually homogenized with 30 strokes in a Potter-Elvehejm homogenizer. Sufficient ethanolic sodium dodecylsulfate (SDS) was then added to obtain 0.2%, and the suspension gently mixed. Insoluble material was removed by centrifugation in a Sorval HB4 rotor (2000 3 g, 10 min) and the supernatant frozen and lyophilized. Samples were stored at 270°C under high purity nitrogen. Authentic ERT, formerly available from Sigma Chemical Co., was either a gift from Donald Melville or isolated from mushrooms by the method of Melville (11). For analysis by HPLC, each lyophilite was suspended in 0.5 ml (pH 7.3) of aqueous 50 µM thiourea and 100 µM choline (to limit thiourylene and quaternary ammonium ion binding to tissue protein) and filtered through a Centricon-10 ultrafiltration unit (Amicon Corp., Beverly, MA). Separation was carried out on two Econosphere C18 columns (Alltech Associates, Deerfield, IL; each column 250 mm 3 4.6 mm, five µ particle size connected in tandem). Several other ODS-type columns tested did not retard ERT satisfactorily. The isocratic elution buffer was 50 mM sodium phosphate, pH 7.3, pumped at ambient temperature and a flow rate of 1 ml/min using a Beckman 110B HPLC solvent delivery module and a 160 Absorbance Detector equipped with 254 nm filters. The concentration of glutathione in each sample was determined by chromatography of a 50 µL aliquot from the resuspended lyophilite of each sample on a Beckman 6300 amino acid analyzer system using glucosaminic acid (Sigma Chemical Co.) as an internal standard. The resolution of ERT from other compounds present in ethanolic extracts of ocular tissues using the HPLC method described is shown in Fig. 2. The method was linear through 10 mM; the practical lower limit of ERT detection was 0.5 µM. The identity of the peak listed as ERT was confirmed by co-chromatography with authentic ERT and by treatment of the sample with trace quantities of I2, which selectively removes 2-thioimidazoles from the sample, followed by rechromatography of the treated sample using the same HPLC system (Fig. 2). RESULTS AND DISCUSSION Recoveries of internal standards averaged $97% for both bovine and porcine aqueous humor and vitreous humor
Ergothioneine in Ocular Tissues
FIG. 2. HPLC Separation of ERT from Retinal Extracts: (A)
preparation of authentic ERT; (B) extract of bovine retina; and (C) extract of porcine vitreous. The arrow indicates the eleution position of ERT. The insert A shows the effect of elemental iodine on the ERT peak (upper tracing: unoxidized, lower tracing: iodine treated).
samples; $95% for pigmented retinal epithelium and retina; $90% for lens; and $87% for cornea. ERT was detected in all ocular tissues examined (Table 1). Bovine lens ERT was present at concentrations about 17- to 25-fold larger than those in cornea and retina, respectively. ERT concentrations in porcine lens were considerably lower than concentrations found in bovine lens. The range of bovine lens value is large (SD about 60% of mean); an explanation for this variation is not apparent. Porcine retina and pigmented retinal epithelium concentrations of ERT were several-fold greater than values in bovine retina and pigmented retinal epithelium and notably greater than
119
lens concentrations. Although the ERT content of pig lens was markedly lower than that of the cow lens, the ERT content in pig erythrocytes was about 53 greater than the ERT concentration in bovine erythrocytes. Erythrocyte ERT concentrations, which are thought to reflect the amount of ERT in the diet at the time of hematopoiesis (5), suggest that overall dietary intake of ERT was approximately 53 larger in pigs than in cows. Concentrations of reduced glutathione (G-SH) were of the same order of magnitude as ERT concentrations in most of the bovine ocular tissues examined (Table 1). Exceptions were bovine lens, where ERT occurred at concentrations about 353 greater than G-SH, and the cornea where ERT concentrations were about 143 greater than those of GSH. In the pig, G-SH and ERT concentrations were similar in the vitreous humor and the aqueous humor, cornea, perhaps the retina and pigmented retinal epithelia, but in the porcine lens G-SH was about 453 greater than ERT concentrations. To assess whether higher ERT levels in the tissues of one species were balanced by lower G-SH concentrations in the same tissues of other species, and vice versa, values for ERT and G-SH of each tissue were summed (5RSH) and the cow/pig ratios of these sums compared (Table 1). While the ratios were relatively constant for four of seven ocular tissues, the large variation (8.45, for instance for the lens) discounted the notion of balance. The role of oxidative mechanisms and antioxidants in the development and control of various pathologies, including those of the eye, is widely recognized. In the lens, a high level of G-SH appears important for maintenance of normal transparency (13). There is an inverse relationship between extent of cataract formation and ERT concentration in the human lens (11). ERT inhibits lipid peroxidation (1,6), hydroxyl radical formation (2,12), singlet oxygen generation (6,14), and the rate of peroxide formation (5). Reactivity of ERT with hydroxy- and α-halogenated peroxy radicals measured by pulse radiolysis is superior to G-SH (7). Given the antioxidant properties of ERT, its presence in ocular tissues at concentrations approximating those of G-SH argues for consideration of this thiourylene as a relevant biological antioxidant mechanism. The cause of the marked species-specific differences in ocular tissue content of ERT is not clear. Gender and age may be discounted, since most animals were male and all about the same age. However, the diet at the time of hematopoiesis is known to affect ERT concentration in the erythrocyte. Porcine concentrations of RBC ERT were greater than bovine levels, suggesting pigs were better provided with dietary ERT than the cattle. Reliable information on the feeding regimen of commercially slaughtered animals is difficult to estimate. Thus, comparison of the level of ERT uptake with tissue levels was not possible. Shukla et al. (15) have reported human lens levels of ERT of about 5 µmoles/ g tissue, a level intermediate between the values we ob-
T. K. Shires et al.
120
TABLE 1. Distribution of ergothioneine and glutathione in tissues of the eye*
Bovine Tissue Aqueous humor** Vitreous humor† Retina† PRE† Lens† Cornea† RBC‡
ERT 1.2 0.12 2.96 0.49 7.79 4.46 0.04
(0.50) (0.01) (0.20) (0.39) (4.72) (1.34) (0.08)
Porcine
GSH
ERT 1 GSH
0.17 (0.05) 0.09 (0.01) 2.41 (0.05) 0.77 (0.05) 2.33 (0.05) 0.31 (0.05) NA
1.37 (0.55) 0.21 (0.01) 5.37 (0.25) 1.26 (0.39) 10.1 (5.99) 4.77 (1.43) NA
ERT 0.76 1.00 8.69 2.97 0.20 0.93 0.22
(0.26) (0.45) (1.57) (1.10) (0.11) (0.48) (0.18)
Ratio
GSH
ERT 1 GSH
Cow/Pig
0.56 (0.18) 0.08 (0.02) 5.28 (0.91) 5.14 (0.47) 9.30 (0.49) 0.56 (0.14) NA
1.32 (1.04) 1.08 (0.19) 14.0 (0.38) 8.11 (0.16) 9.50 (8.45) 1.49 (3.20) NA
1.04 0.19 0.38 0.16 1.06 3.20 NA
*Values shown are means from six steers or nine pigs with standard deviations shown in parentheses. ** µmoles/mL. † µmoles/gm tissue. ‡ µmoles/gm RBC. NA 5 not analyzed. PRE 5 pigmented retinal epithelium.
served for cows and pigs. Until more is known about the mechanism of ERT uptake and distribution to tissues, the significance of the differences in tissue levels will remain obscure. Supported in part by the Logan Research Fund, Research to Prevent Blindness; a grant from the Mead Johnson Nutritional Group; and by the Children’s Miracle Network Telethon. The assistance of Heather Jackson and Gwen Knapp is gratefully acknowledged, as is the help of Mel and Neva Herschberger of the Kalona Packing Plant, Kalona, IA.
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