- Email: [email protected]
In search of a physiological function for L-ergothioneine
Medical Hypotheses18: 351-370,1985
IN SEARCH OF A PHYSIOLOGICAL FUNCTION FOR L-ERGOTHIONEINE M.C. Brummel, Pharmaceutical Development Systems, 2920 Industrial Park Road, Iowa City, Iowa 52240 ABSTRACT Since its discovery at the turn of the century, attempts to define a physiological Eunction for This paper suqgests L-ergothioneine have been unsuccessful. several possible functions for this enigmatic compound or transport of cations or These include: its metabolites. carbon dioxide, catalysis of carboxylation or decarboxylation reactions, mediation of .thyroid or antithyroid Eunction, histaminic or antihistaminic action, and cholinergic or anticholinqeric action. INTRODUCTION Ergothioneine is the trimethylbetaine of 2-thiohistidine (Fig 1) synthesized in the lower eukaryotes Neurospora crassa and Claviceps purpurea from a histidine backbone, a cysteine sulfur and 3 S-adenosyl methionyl methyls and not presently believed to be synthesized in higher eukaryotes in which levels appear to be a reflection of dietary ergothioneine.
COOH I
, CHPCH
HC= I N PC/ Figure 1.
BH
L-Ergothioneine 351
’ CHjbH, I bH, NH
An excellent review of ergothioneine literature has been published by Melville (1) with only limited additional studies being published since that time. To better understand this compound and its literature, it is necessary to deal with its quantitative determination. In early literature, ergothioneine measurement tias complicated by the lack of specificity of procedure or the lack of satisfactory recovery of standard ergothioneine. The development by Hunter (2,3) of a diazotization procedure to yield magenta-colored compounds (Fig. 2A) which require an unsubstituted sulfur and imidazoyl carbon added credibility to ergothioneine determinations, however, interferring substances and the possibility of various side chains off the thioimidazole skeleton leave doubt as to what was actually being measured. Joceyln (4) developed an assay based on the base-catalyzed elimination of trimethyl amine from ergothioneine or ergothioneine-like compounts (Fig. 2b). This assay is complementary to the Hunter method and nonspeciEic for imidazoyl derivatization. The naturally occuring methylated quaternary ammonium compounds; choline, carnitine, and betaine, are negative in this test, while lecithin was not studied. R
,coo”
F--H2&,,i N\ v NH -
I-_-_--J
3:
Figure 2. A - Colored product of diazotization ergothioneine in the Hunter assay.
of
B - Basis of Joceyln assay of ergothioneine-like compounds by release of trimethylamine.
352
By trimethylamine release assay, human plasma and plasma proteins have been found to contain 1.0 to 2.6 mg "erqothioneine" per 100 mls. (4). The diazo method qives a plasma concentration of zero to about 1 mq/lOO ml, but this is not considered to be erqothioneine (5,6,7). The implication is that plasma and its proteins contain an ergothioneine-like compound. It has also been observed that other sulphur containins erqothioneine-like compounds arr? found in serum (8) and semen (9,lO). One such compound is hercynine (histidine betaine), which has been found in blood (11) and can be produced by the oxidative desulphur.ation of ergothioneine (Fig. 3).
,cos
r-f
CH,C\H
-
,+,N(CH4
02
Figure 3. Formation of hercynine by the facile oxidative desulphuration of ergothioneine. Unlike the potentially interferring nonergothioneine thiols and ascorbic acid Found in blood, ergothioneine sulfhydryl groups are stable to Cu++ catalyzed alkaline oxidation. This has been used as a method for erqothioneino ,determination using 2, 2'-dipyridyl disulphide, (12,131 yielding values generally in aqreenent with the lower yrrj:fues obtained by the diazo or trimethylamine release assays. Interestingly, acid precipitation of blood proteins in the 'absence of added thiols causes the lnss oE diazo-posF?ivc~: serqothioneine (14) and the loss of 2,2'-dipyridyl disulphide-positive added ergothioneine. (13) 'This is consistent with oxidative desulfuration caused by oxygrn release Erom denatured oxyhemoglobin. (15) Most recently, more specific HPLC (16) and TLC ,171 methods for erqothioneine ,assay have confirmed the pr't~+:~c~ of erqothoineine in rat Liver, kidney, testicle, heart, spleen, lung, urine, feces, brain and plasma, indicating it may be ubiquitous in animal_ tissues. The general picture emergent from the preceding is !:hat we cannot be sure what is beinq measured in the experiment5 using calorimetric assays and that there exist
353
ergothioneine-like compounds which may be artifacts of the isolation procedure. The apparent ease in finding nonergothioneine but ergothioneine-like compounds might suggest that ergothioneine itself is not the physiologically active form of the compound. DISCUSSION Does erqothioneine have a carnitine-like the pentose phosphate pathway?
function related to
A molecular similarity exists between carnitine and ergothioneine (Fig. 4). Carnitine has a quaternary ammonium cationic charge separated from a carboxylate anionic charge by 3 carbon-carbon bonds; the central carbon bearing the hydroxyl function which is esterified with fatty acids for transport across the mitochondrial membrane. Analogously, ergothioneine has the same quarternary ammonium/carboxylate functions only they are separated by a single carbon from which a thioimidazole "ligand" extends. It is therefore possible that ergothioneine "shuttles" carbon dioxide (bound to imidazole nitrogens), protons, mono or divalent cations (bound to the sulfur), electrons (reduced form of thioimidazole ring), or acyl groups (bound by a thioester bond similar to that found in acetyl CoA or acetyl lipoate).
I I
CH,
01
/p
I
CHsN / I ‘\ /’ c’ CH:\ 4’ ) %I i___________CH________A ERGOfHIONEINE L* h
N, NH Y
SH
Figure 4. Ergothioneine and carnitine, a known transport molecule, have areas of molecular similarity.
354
Carnitine is located in the mitochondrial membrane where it shuttles lipid-derived long-chain fatty acids into the mitochondria where, by p-elimination, the acetate needed Carnitine also for fueling the Krebs cycle is formed. transports acetyl CoA from the inside of the mitochondria to the outside cytosol where long-chain fatty acid synthesis leading to lipid formation occurs. (18) Ergothioneine has also been found in a bound form in a membrane fraction from rat liver cells. (19) In red blood cells an active and very important pentose phosphate pathway supplies the mitochondrionless (no respiratory chain) cell with metabolic energy (ATP) through anaerobic glycolysis with Eormation of lactic acid. Interestingly, ergothioneine added to either suspended or hemolyzed rat red blood cells accelerates lactate formation and decreases glucose-6-phosphate and fructose-6-phosphate without altering pyruvate levels. (20) Ergothioneine adininistration to intact rats also prevented a 40% stravation-induced diminution of red blood ceLL lactate seen in controL animals. (20) Since ergothioneine has been shown to sensitize certain ribosidases to inhibition by nicotinamide, blocking phosphorolytic cleavage of NAD+ and NADP+, (21,221 ergothioneine may regulate the conversion of glucose-6-phosphate to 6-phosphoglucona lactone by controlling the NAD+ required for the pyruvate to lactate conversion efEected by lactate dehydrogenase in anaerobic gLycoLysis. PlatoLets have an active pentose phosphate pathway whic:h i.s the chief source of CO2 produced from glucose. (23) They are also responsible for 25-308 of the total Lipid synthesis per unit of whole blood. (24) It would therefore appear that synthesis of acetyL CoA Erom pyruvate oxidized via the Krebs cycle must be important. Roth carnitine and eriJothioneine added in vitro to human platelets increase the i,roduction of CO2 from pyruvate, acetate, or 3-hydroxybutyrate. (25) For both compounds this occurs withouL_ increase in oxygen consumption indicating the effect ir, r!-lated to fatty acid metabolism and not respiration. T,iver, kidney, and red blood ceLLs aLl of which have significant pentose phosphate pathways also have been found tl, contain the highest amounts of ergothioneine. Frgothioneine may al50 have d respiratory effect in !;:l)'mC? in,;tances as it doubles the survival time of gllines pig ::;)ermatozoaMhile accelerating their rate of respiration with fructose a:; substrate and relieving the respiratory inhibition produced by P-chloro,ner._uribenzoat~. (261 If ergothioneine turns on a pentose phosphate pathway to sllpply ATP by anaerobic ylycolysis, a cell which contains 00 respiratory chain (erythrocyt_e) or a cel.1 which has had 355
no respiratory chain poisoned by cyanide, nitrite, or sulphydryl inhibitors might be expected to better survive the energy demand if ergothioneine content is augmented. Supporting such a function for ergothioneine are the observations that ergothioneine is antidotal to cyanide administration in the rat (27); protects from inhibition by sulphyaryl inhibitors the motility and aerobic and anaerobic fructolysis of spermatozoa (26,28); relieves the inhibition of anaerobic, but not aerobic, fructolysis in spermatozoa which is caused by Cu++ or H202 (28); and relieves the inhibition of lactate formation by sulphydryl inhibitors in Meyerhof extracts of rabbit skeletal muscle and alcoholic baker's yeast fermentations. (28) In red blood cells and liver cells, glutathione must be kept in the reduced form, a process requiring NADPH generated from the pentose phosphate pathway. The reduced glutathione is needed to convert methemoglobin, the Fe+++ nonoxygen binding form of hemoglobin, into Fe++ hemoglobin, the oxygen binding form. A cell or tissue in which ample ergothioneine insures an active pentose phosphate pathway would be better able to withstand stresses directed toward converting Fe++ to Fe+++ or not converting Fe+++ to Fe++. Nitrite ion causes such a stress and the resultant methemoglobinemia produced by ingestion of nitrate-contaminated well water is seen only in very young infants. (29) This may be because in both humans (30) and rats (31) blood ergothioneine levels are found to be lower in infants than in adults. (32) It is of more than passing interest to note the existence of another quaternary ammonium derivative of histidine. Post-translational modification of a histidine residue in the primary sequence of eukaryotic elongation factor-2 results in formation of diphthamide. When this residue is exposed to diptheria toxin, one of the imidazoyl nitrogens is ADP-ribosylated. (33,34,35) Ert may be open to similar ribosylation, either through an imidazoyl nitrogen or through a thioribosyl linkage at position 2 of the imidazoyl ring. Since Ert has been found in protein-bound form and since similar thiourylenes (the antithyroid methimazole and the antineoplastic 6-mercaptopurine) can be covalently attached to proteins (36,371, one can speculate that the expression of the physiological function of ergothioneine may be inhibited by diptheria toxin through ADP-ribosylation. Is erqothioneine
involved in cation transport?
Sulfur atoms resemble oxygen atoms when viewed using the Periodic Law. Oxygen has a 1~2 2s2 2~4 electronic configuration while sulfur has a 1~2 2~2 2~6 3~4
356
Since p-ofbitals require 6 electrons to be configuration. filled, both oxygen and sulfur can participate in two Because of its greater number of inner covalent bonds. shell electrons, sulfur is considerably less electronegative than oxygen and tends to form two single bonds where its oxygen analogues would form one double bond. In ergothioneine, the sulfur attached to the imidazole ring should be capable of binding and transporting monovalent cations such a H+, Na+, and K+ in a 1 to 1 molar ratio or divalent cations such as Cu++, Zn++, Ca++, or Mgtt using two ergothioneine per cation. Ergothioneine-monovalent cation complexes have not been investigated. Ergothioneine-divalent cation complexes have been formed, and 2 ergothioneine molecules are found to form extremely stable complexes with single atoms of Cu++ > Hq++> Zn++ > Cd++ > Co++ = Ni+t. (38, 39) Unfortunatley, Ca++-and Mg++ complexes were not studied. Since the administration of ergothioneine has been found to prevent cadium-induced teratogenesis in mice (40) it may be that poisonings caused by this and other metals may result from blocking the normal physiological function of endogenous ergothioneine. Epand (41) suggested that dietary ergothioneine may The play a role in the development of diabetes mellitus. reasoning is that ergothioneine has been found to be elevated in the blood of some diabetics. (42) Since ergothioneine levels have been found to have a strong affinity for Zn++, 138,391 and since Zn++ is important Ear the storage and secretion of insulin and glucagon from pancreatic islet cells, (43) perhaps this elevated ergothioneine is removing needed Zn++ and with it normal pancreatic reponse to glucose. Alternatively, abnormal diabetic pancreatic secretory activity may give rise to elevated blood ergothioneine levels. Interestingly, the removal of the rat pancreas produces a marked decrease in red blood cell ergothioneine. (44) Certain divalent metal-ion chelators are known to be diabetogenic compounds. Among these are certain analogues of 8-aminoquinoline, (43) the diabetogenic action paralleling the strength of the quinoline-Zn++ complex. Red blood cells deficient in glucose-6-phosphate dehydrogenase, an important pentose phosphate pathway enzyme, are more susceptible to the toxic action of certain 8-amino-quinolines. The question arises, does the physiological function of ergothioneine require Zn++ for expression? Like cancer, diabetes should be thought of as a malfunctioning process composed of many interrelating actions and reactions, any one of which may malfunction to produce the diabetic state. 357
Ergothioneine may be involved in Zn++ transport into cells. As an example, the red blood cell appears resistant to exchange with exogenous ergothioneine leading to the speculation that ergothioneine incorporation occurs only during erythropoesis. (1) Before maturation of red blood cells, at which time protein synthesis is terminated, large amounts (1 mg/ml packed cells) of the Zn++ metalloenzyme Does ergothioneine carbonic anhydrase must be synthesized. facilitate the Zn++ acquisition process? Does ergothioneine serve as a mediator for carbon dioxide fixation by carbonic Might similar ergothioneine Zn++-enzyme anhydrase? situations occurs in other cells? The reverse action of ergothioneine, the removal of Zn++ from Zn++ metalloenzymes, cannot be a general phenomena since the Zn++ metalloenzymes alcohol dehydrogenase, glutamate dehydrogenase and alkaline phosphatase are not inhibited by the addition of ergothioneine. (38) The capacity to take up, store, and decarboxylate amines may be quite widespread among polypeptide-secreting endocrine cells. (45) Perhaps ergothioneine plays a role in facilitating CO2 transfer in decarhoxylation reactions. Ergothioneine may be a cofactor decarboxylation reactions.
in carboxylation
or
The nitrogen atoms of a 2-thioimidazole are more It is nucleophilic than on the analogous 2-oxoimidazole. already known that the cofactor biotin, which contains an oxygen bearing ring, is involved in carbon dioxide Eixstion reactions, such as those catalyzed by the carboxylases for pyruvate, acetyl CoA, and propionyl Coil as well as Eor An immediate is methylmalonyl transcarboxylase. biotin-bound carbon dioxide (Fig. 5). One mig!lt suspect that ergothioneine would be even better at such a function. There is no experimental evidence to date which would indicate such a function, however, certain ergothioneine-like compounds with blocked nitrogens have been demonstrated to be antithyroids which may interEere with normal CO2 "transport" by ergothioneine.
358
“co;’ z ATP
r--
!
,c-o“co, E
r
_
t
----
CH~CH PROTEiN I +NlMel, ___-
2
;
ATP?
SH
I
,________-_-1
I _________---A
ERGOTHIONEINE
Figure 5. The thioimidazole bind carbon dioxide in carboxylation reactions in a manner similar to Thyroid or antithyroid ergothioneine.
nitrogens of ergothioneine may carbon dioxide transport, or decarboxylation reactions, that observed for biotin.
drugs may act on a process
involving
An antithyroid will decrease the basal metabolic rate This is measured directly by heat output or of an organism. indirectly by oxygen consumption or CO2 elimination. Various antithyroid drugs (Fig. 6) have a thiocarbamide structure suggesting a possible interference in an In addition, carnitine, ergothioneine mediated pathway. already seen to have certain ergothioneine-like molecular similarity, is an antithyroid. Initial experiments in which 50g female rats were given 2 mg/kg/day ergothioneine by injection over ten days followed by removal of their thyroids and assay of. iodincontent indicated ergothioneine and 2-thiouracil-treated rat thyroids had about 10% of the iodine contained in control thyroids. (.46) Subsequent studies in animals including humans failed to reproduce this initial observation. (47-311
359
r-f
II
I
Nwe
Nv
CH,CH$H,
CH,CH&H, I,,,,,-,-------
SH
Figure 6. There is an area of structural homology between ergothioneine and certain antithyroid drugs including A methimazole l3 propylthioimidazole (3 carbimazole D propylthiouracil
It would seem informative to check Nl-methyl, Nl-carbethoxy, or the g isomer of ergothioneine for thyroid or antithyroid activity. Additionally, if ergothioneine plays a role in both thyroid-antithyroid function and the diabetic condition, one might expect diabetics to have altered basal metabolism. Many diabetics are obese. Antithyroids are known to cause agranulocytosis. If these drugs are antiergothioneinics, granulocytes might be expected to contain ergothioneine or metabolites of ergothioneine. IE ergothioneine is incorporated into white blood cells only during early differentiation stages, one would expect to find ergothioneine in the bone marrow. This has been demonstrated. (52)
360
Ergothioneine or its derivatives, antihistaminic.
may be histaminic
or
Histidine is Ergothioneine is a histidine derivative. not pharmacologically active, but can be made so by histidine decarboxylase which liberates carbon dioxide and histamine, the latter being a very important and versatile molecule. Might ergothioneine suffer a similar fate, notably decarboxylation to a vasoactive amine (Fig. 7)3
/COO
l-l
CH,CH \ @NH,
/COO
\ V
N
0
HDC e
l-f
CH,CH, \ ,+NH,
G-3
CH,CH \ BN(Me),
CH,CH, \ ,,#(Me),
EDC?
NH
ERGOTHIONEINE
r-f
CH&$i aN(Me),
z
HERCYNINE
B
F'igur3 7. Two possible fates of ergothioneine metabolism. 4 - 'The formation of trimethyl thiohistamine. 3 - The Formation of trimethylhistaminr?. 361
Hercynine could be generated by oxidative desulfation. Subsequent decarboxylation would yield K-N-trimethylhistamine. Alternatively, decarboxylation of ergothioneine could Eorm Z-thio-Cd-N-trimethyl) histamine, which may OK may not be further metabolized. Hercynine has been found in blood (13.). Trimethyl histamine or trimethyl thiohistamine have not been knowingly assayed but may have been measured unknowingly in certain assays for histamine or ergothioneine. Similar compounds have been tested for pharmacological activity (Table 1). (53) Observe that&-N-methyl and "-N-dimethyl histamine have two to three times the gastric acid secretion activity as the same amount of histamine. Also observe that 2-thio-histamine has virtually no histaminic activity on smooth muscle Hl receptors. Methylating the imidazoyl nitrogens of histamine inactivates both HI and gastric acid secretion activites. Synthesis and testing of the compounds indicated should prove exceedingly informative.
R’
R
R”
R//f
Hl
H2
I --
H
H
1.0
1.0
HzCH3
--
H
H
1.0
2.5-3.0
WCH312
--
H
H
0.8
2-3
-_
H
0.006
H3
.R
CH.jZH,N
i-f R’
.N
vNxR” ’ ,,I R
/ Compounds of interest not tested
i
H3
CH3
H3
--
CH3
H3
--
H
SH
H(CH3)3
--
H
H
H(CH3j3
--
H
SH
--
H
SH
H3
TABLE 1
362
H
0
0 0
0.0005
? 0.0005
7
An example: Suppose a Interesting hypotheses emerge. cell carrying ergothioneine (eg-eosinophill) arrives at an inflammation site with the intent of controlling an inflammatory reaction through an antihistaminic pathway. Ergothioneine could be decarboxylated and the resultant molecule donates a methyl group to the imidazoyl nitroqen of any available histamine or vasoactive amine resulting in the Further suppose formation of two inactive compounds. Enter superoxide anion or modulation is not yet desirable. myeloperioxidase resulting in oxidative desulfuration of any histamines which thioimidazole, formingd-N-methylated Consider what may happen augment the histaminic response. under abnormal conditions. Trial and error experiments for antihistaminic compounds resulted in the development of Burimamide, Metiamide, and Cimetidine as specific H2 antogonists (Fig. 8). A thiocarbamide-like structure is found in each of these molecules and one wonders if these areas interfere with normal ergothioneine or egothioneine-like functions. One also wonders if closing the ring on these compounds (Fig. 8B) would give rise to more effective (specific and potent) H2 antagonists.
yc
CH,S II
kH2
l-f
I
CH,
I
“Wb.,,,,,, “V”” I HNCN
HNCN
I I
A
Figure 8.
6
A-Known H2 antagonists B-Potential H2 antagonists
A known cholinergic ergothioneine.
has structural
not yet tested
homolgy with
Pilocarpine, a cholinergic, has structural homology with ergothioneine (Fig. 9). Ergothioneine also has The obvious implication structural homology with choline.
363
is that ergothioneine, or a derivative of ergothioneine, might have cholinergic or anticholinergic action. Alternatively, ergothioneine or some derivative of ergothioneine may have an important function similar to but independent of the choline-acetylcholine system.
H
r-----
’
Me
/MeN” Me+ 8 (
01 6~ 1t-h
L____-_+----_
H CHOLINE
r_--_---__4
I
01
Me
61
/ MeN”
, Me+ i______+____,
eo
FH? - tRGoT”IONtlNtNtl=l ti
v\
/
/I hH
SH
Figure 9. Ergothioneine bears areas of structural homolgoy with choline/acetylcholine and the cholinergic drug pilocarpine. Ergothioneine has been reported to be exictatory on the electrical activity of the cerebellum of the decerebrate rabbit. (54) Ergohtioneine can also have excitatory effects on neurons in the brainstem. (55) Other reports dispute these findings. (56,571 CONCLUSIONS Presented are some easily testable hypotheses on possible physiological Eunctions for ergothioneine. It is important to keep in mind that ergothioneine itself may be inactive ant that it may be converted into an active form(s).
364
There exists a tantalizing possibility that ergothioneine may have different and even opposite physiological functions dependent on the route of its metabolism. It seems likely that within the framework of the hypotheses presented, a physiological function Ear ergothioneine will emerge.
365
REFERENCES 1.
Melville
DB.
2.
Hunter G. A new test for ergothioneine upon which is based a method for its estimation in simple solution and in blood filtrates. Biochem J, 1928;22:4.
3.
Hunter G. Colour reactions of thioglyoxalines (thioimidazoles) with sodium diazobenzene-p-sulphonate. J Chem Sot, 1930;2343.
4.
Jocelyn PC. The distribution of ergothioneine in blood as determined by a new method of estimation. Biochem J, 8;70:656.
5.
Hunter G. The determination of ergothioneine in simple solution and in blood. Can J Res, 1949;27:230.
6.
Fraser R. Blood ergothioneine levels in diabetes mellitus. J Lab Clin Med, 1950;35:960.
7.
Fraser R. Blood ergothioneine Clin Med, 1951;37:199.
8.
Smith EL, Tuller EF. The paper chromatographic detection of the free sulfur-containing amino acids and small peptides in whole blood and serum. Arch Biochem Biophys, 1954;54:114.
9.
Mann T, Leone E, Polge C. The composition of the stallions semen. J Endocrinol, 1956;13:279.
Ergothioneine.
Vitam Horm, 1959;17:155.
levels in diease.
J Lab
10.
Heath H, Rimington C. Further studies on seminal ergothioneine of the pig. Biochem J, 1957;65:369.
11.
Ackerman D, List PH, Menssen HG. Uber das vorkommen von herzynin neben ergothioneine in der samen-flussigkeit des ebers sowie in rinder erythrocyten und die biologische beziehung der beiden basen zueinander. 2 Physiol Chem, Hoppe Seyler's,1959;314:33.
12.
Carlsson J, Kierstan MP, Brocklehurst K. Reactions 1-ergothioneine and some other aminothiones with 2,2'-and 4,4 '-dipyridyl disulphides and of 1-ergothioneine with iodoacetamide. Biochem J,1974;139:221. 366
of
13.
Carlsson J, Kierstan MP, Brocklehurst K. A convenient spectrophotometric assay for the determination of l-ergothioneine in blood. Biochem J, 1974;139:237.
14.
Melville DB, Lubschez R. A method for the determination of ergothioneine in blood. J Biol Chem, 1953;200:275.
15.
Heath H, Toennies G. The preparation and properties ergothioneine disulphide. Biochem J, 1958;68:204.
16.
Mayumi T, Kawano H, Sakamoto Y, Suehisa E, Kawia Y, Hama 'I'. Studies on ergothioneine. V. Determination by high performance liquid chromatography and application to metabolic research. Chem Pharm BuLl, 1978;26:3772.
17.
Kaneko I, Takeuchi Y, Yamaoko Y, Tanaka Y, Fukuda T, Fukumori Y, Mayumi T, Hama T. Quantitative determination of ergothioneine in plasma and tissues by tic-densitometry. Chem Pharm Bull, 1980;28:3093.
18.
Fritz IB. Carnitine and its role in fatty acid metabolism. Adv Lipid Res, 1963;1:285.
19.
of
Kawano H, Otani M, Takeyama K, Kawai Y, Mayumi T, Hama Studies on ergothioneine. VI. Distribution and fluctuations of ergothioneine in rats. Chem Pharm BuLL, 1982;30:1760.
T.
20.
Yawano H, Higuchi F, Mayumi T, Hama T. Studies on s?rgothioneine. VII. Some eEfects of orgothioneine on gL:ycolytic metabolism in red blood celLs from rats. IChernPharm Bull, L982;30:2611.
21.
Grossman L, Kaplan NO. Nicotinamide riboside phosphorylase from human erythrocytes. I. ?hosFhor,Jlytic activity. J BioL Chem, 1958;231:717.
22.
Grossman L, Kaplan NO. Nicotinamide riboaide phosph0ryLas.e Erom human erythrocytes. II. Uicotinamide sensitivity. J Biol Chem,1958;23:727.
23.
Yerby GP, Taylor SM. The effect of added ATP on the :lathways of glucose utiLization by human washed ?L.atf+Letsin vitro. &J Lab CLin Med, 1967;69:194.
24.
tiar'isPA, Gelliorn A, Kidson C. Lipid synthesis in J Biol hAnan leukocytes, platelets, and urvthrocytes. [Chem, 1369;235:2573.
367
25.
Kerby GP, Taylor SM. Effect of carnitine and ergothioneine on human platelet metabolism. Proc Sot Exper Biol Med, 1969;132:435.
26.
Hama T, Okumura H, Tamaki N, Konishi T. Studies on ergothioneine. IV. Effect of ergothioneine upon spermatozoa from guinea pigs. Yakugaki Zasshi,1973;93:369.
27.
Trabucchi E. Richerche farmacologiche sulla ergothioneine. Boll Sot Ital Biol Sper, 1936;11:117.
28.
Mann T, Leone E. Studies on the metabolism of semen. VIII. ergothioneine as a normal constituent of boar seminal plasma. Purification and crystallization. Site of formation and function. Biochem J, 1953;53:140.
29.
Comly HH. Cyanosis in infants caused by nitrates well water. JAMA, 1945;129:112.
30.
Touster 0, Yarbro MC. The ergothioneine content of human erythrocytes: the effect of age, race, malignancy, and pregnancy. J Lab Clin Med, 1952;39:720.
31.
Mackenzie JB, Mackensie CG. The effects of age, sex, and androgen on blood ergothioneine. J Biol Chem,1957;225:651.
32.
Spicer SS, Wooley JG, Kessler V. Ergothioneine depletion in rabbit erythrocytes and its effect on Proc Sot Exper methemoglobin formation and reversion. Biol Med, 1951;77:418.
33.
Van Ness BG, Howard JB, Bodley JW. ADP-ribosylation elongation factor 2 by diptheria toxin. J Biol Chem,1980;255:10710.
of
34.
Van Ness BG, Howard JB, Bodley JW. ADP-ribosylation elongation Eactor 2 by diptheria toxin. J Biol Chem,1980;255:10717.
of
35.
Bodley JW, Van Ness BG, Howard JB. In: Smolson M, Sugimura T, eds. Novel ADP-ribosylations of regulatory enzymes and proteins. New York: Elsevier-North Holland, 1980;413-423.
368
in
36.
Hyslop RM, Jardine I. Metabolism of 6-thiopurines. Covalent binding of a 6-thiopurine metabolite to mammalian tissue protein in vivo. J Pharmacol Exp Ther,1981;218:629.
II.
37.
Poulsen LL, Hyslop RM, Ziegler DM. S-oxygenation of thioureylenes catalyzed by a microsomal flavoprotein mixed-function oxidase. Biochem Pharmaco1,1974;23:3431.
38.
Interaction Hanlon, DP. ions and metalloenzymes.
39.
Motohashi N, Mori I, Sugiura Y, Tanaka H. Metal complexes of ergothioneine.Chem Pharm Bull, 1974;22:654.
40.
Mayumi T, Okamoto K, Yoshida K, Kawai Y, Kawano H, Hama T, Tanaka K. Studies on ergothioneine. VIII. Preventive effects of ergothioneine .on cadmium-induced teratogenesis. Chem Pharm Bull, 1982;30:2141.
41.
Epand RM. The role of dietary ergothioneine development of diabetes mellitus. Med Hypotheses,1982;9:207.
42.
Fraser R. mellitus.
43.
Lazaris JA, Bavelskyi ZE. Primary insulin insufficiency after blocking of zinc in pancreatic beta-cells. Endocrinol Exp (Bratisl), 1981;15:99.
44.
Beatty CH. Levels of ergothioneine in the red blood cell in experimental diabetes. J Biol Chem,1952;199:553.
45.
Pearse AGE. Mem Sot Endocrinol,
46.
Lawson A, Rimington C. Antithyroid activity ergothioneine. Lancet, 1947;252:586.
47.
Searle CE, Lawson A, Hemmings AW. Antithyroid substances. I. The mercaptoglyoxalines. Biochem J,1950;47:77.
of ergothioneine with metal J Med Chem, 1971;14:1084.
in the
Blood ergothioneine levels in diabetes J Lab Clin Med, 1950;35:960.
369
1971;19:543. of
48.
Heath H, Rimington C, Searle CE, Lawson A. Some effects of administering ergothioneine to rats. Biochem J, 1952;50:530.
49.
Baldridge RC. Blood ergothioneine Nutrition, 1955;56:107.
50.
Wilson ML, McGinty DA. Antithyroid activity ergothioneine. Am J Physiol, 1949;156:377.
51.
Astwood EB, Stanley MM. Antithyroid activity ergothioneine. Lancet, 1947;252:586.
52.
Lutwak-Mann C. in bone marrow.
53.
Histamines and anti-histamines. Part II. M Kocha e Silva, ed. Berlin: Springer-Verlag, 1966;182:427.
54.
Crossland J, Mitchell JF, WoodrufE GN. The presence ot ergothioneine in the central nervous system and its probable identity with the cerebellar factor. ,J Physiol, 1966;182:427.
55.
Avanzino GL, Bradley PB, Comis SD, WolstencroEt JH. A comparision of the actions oE ergothioneine and chlorpromazine applied to single neurons by two different methods. Int J Neuropharmacol, 1966;5:331.
56.
Krnjeric K, Randic M, Straughan DW. Ergothioneine central neurons. Nature, 1965;205:603.
57.
Briggs I. Ergothioneine in the central nervous systems. J Neurochem, 1972;19:27.
and dietary oats.
Some aspects of phosphorous Biochem J, 1952;52:356.
370
,I
of
of
metabolism
and
IN SEARCH OF A PHYSIOLOGICAL FUNCTION FOR L-ERGOTHIONEINE M.C. Brummel, Pharmaceutical Development Systems, 2920 Industrial Park Road, Iowa City, Iowa 52240 ABSTRACT Since its discovery at the turn of the century, attempts to define a physiological Eunction for This paper suqgests L-ergothioneine have been unsuccessful. several possible functions for this enigmatic compound or transport of cations or These include: its metabolites. carbon dioxide, catalysis of carboxylation or decarboxylation reactions, mediation of .thyroid or antithyroid Eunction, histaminic or antihistaminic action, and cholinergic or anticholinqeric action. INTRODUCTION Ergothioneine is the trimethylbetaine of 2-thiohistidine (Fig 1) synthesized in the lower eukaryotes Neurospora crassa and Claviceps purpurea from a histidine backbone, a cysteine sulfur and 3 S-adenosyl methionyl methyls and not presently believed to be synthesized in higher eukaryotes in which levels appear to be a reflection of dietary ergothioneine.
COOH I
, CHPCH
HC= I N PC/ Figure 1.
BH
L-Ergothioneine 351
’ CHjbH, I bH, NH
An excellent review of ergothioneine literature has been published by Melville (1) with only limited additional studies being published since that time. To better understand this compound and its literature, it is necessary to deal with its quantitative determination. In early literature, ergothioneine measurement tias complicated by the lack of specificity of procedure or the lack of satisfactory recovery of standard ergothioneine. The development by Hunter (2,3) of a diazotization procedure to yield magenta-colored compounds (Fig. 2A) which require an unsubstituted sulfur and imidazoyl carbon added credibility to ergothioneine determinations, however, interferring substances and the possibility of various side chains off the thioimidazole skeleton leave doubt as to what was actually being measured. Joceyln (4) developed an assay based on the base-catalyzed elimination of trimethyl amine from ergothioneine or ergothioneine-like compounts (Fig. 2b). This assay is complementary to the Hunter method and nonspeciEic for imidazoyl derivatization. The naturally occuring methylated quaternary ammonium compounds; choline, carnitine, and betaine, are negative in this test, while lecithin was not studied. R
,coo”
F--H2&,,i N\ v NH -
I-_-_--J
3:
Figure 2. A - Colored product of diazotization ergothioneine in the Hunter assay.
of
B - Basis of Joceyln assay of ergothioneine-like compounds by release of trimethylamine.
352
By trimethylamine release assay, human plasma and plasma proteins have been found to contain 1.0 to 2.6 mg "erqothioneine" per 100 mls. (4). The diazo method qives a plasma concentration of zero to about 1 mq/lOO ml, but this is not considered to be erqothioneine (5,6,7). The implication is that plasma and its proteins contain an ergothioneine-like compound. It has also been observed that other sulphur containins erqothioneine-like compounds arr? found in serum (8) and semen (9,lO). One such compound is hercynine (histidine betaine), which has been found in blood (11) and can be produced by the oxidative desulphur.ation of ergothioneine (Fig. 3).
,cos
r-f
CH,C\H
-
,+,N(CH4
02
Figure 3. Formation of hercynine by the facile oxidative desulphuration of ergothioneine. Unlike the potentially interferring nonergothioneine thiols and ascorbic acid Found in blood, ergothioneine sulfhydryl groups are stable to Cu++ catalyzed alkaline oxidation. This has been used as a method for erqothioneino ,determination using 2, 2'-dipyridyl disulphide, (12,131 yielding values generally in aqreenent with the lower yrrj:fues obtained by the diazo or trimethylamine release assays. Interestingly, acid precipitation of blood proteins in the 'absence of added thiols causes the lnss oE diazo-posF?ivc~: serqothioneine (14) and the loss of 2,2'-dipyridyl disulphide-positive added ergothioneine. (13) 'This is consistent with oxidative desulfuration caused by oxygrn release Erom denatured oxyhemoglobin. (15) Most recently, more specific HPLC (16) and TLC ,171 methods for erqothioneine ,assay have confirmed the pr't~+:~c~ of erqothoineine in rat Liver, kidney, testicle, heart, spleen, lung, urine, feces, brain and plasma, indicating it may be ubiquitous in animal_ tissues. The general picture emergent from the preceding is !:hat we cannot be sure what is beinq measured in the experiment5 using calorimetric assays and that there exist
353
ergothioneine-like compounds which may be artifacts of the isolation procedure. The apparent ease in finding nonergothioneine but ergothioneine-like compounds might suggest that ergothioneine itself is not the physiologically active form of the compound. DISCUSSION Does erqothioneine have a carnitine-like the pentose phosphate pathway?
function related to
A molecular similarity exists between carnitine and ergothioneine (Fig. 4). Carnitine has a quaternary ammonium cationic charge separated from a carboxylate anionic charge by 3 carbon-carbon bonds; the central carbon bearing the hydroxyl function which is esterified with fatty acids for transport across the mitochondrial membrane. Analogously, ergothioneine has the same quarternary ammonium/carboxylate functions only they are separated by a single carbon from which a thioimidazole "ligand" extends. It is therefore possible that ergothioneine "shuttles" carbon dioxide (bound to imidazole nitrogens), protons, mono or divalent cations (bound to the sulfur), electrons (reduced form of thioimidazole ring), or acyl groups (bound by a thioester bond similar to that found in acetyl CoA or acetyl lipoate).
I I
CH,
01
/p
I
CHsN / I ‘\ /’ c’ CH:\ 4’ ) %I i___________CH________A ERGOfHIONEINE L* h
N, NH Y
SH
Figure 4. Ergothioneine and carnitine, a known transport molecule, have areas of molecular similarity.
354
Carnitine is located in the mitochondrial membrane where it shuttles lipid-derived long-chain fatty acids into the mitochondria where, by p-elimination, the acetate needed Carnitine also for fueling the Krebs cycle is formed. transports acetyl CoA from the inside of the mitochondria to the outside cytosol where long-chain fatty acid synthesis leading to lipid formation occurs. (18) Ergothioneine has also been found in a bound form in a membrane fraction from rat liver cells. (19) In red blood cells an active and very important pentose phosphate pathway supplies the mitochondrionless (no respiratory chain) cell with metabolic energy (ATP) through anaerobic glycolysis with Eormation of lactic acid. Interestingly, ergothioneine added to either suspended or hemolyzed rat red blood cells accelerates lactate formation and decreases glucose-6-phosphate and fructose-6-phosphate without altering pyruvate levels. (20) Ergothioneine adininistration to intact rats also prevented a 40% stravation-induced diminution of red blood ceLL lactate seen in controL animals. (20) Since ergothioneine has been shown to sensitize certain ribosidases to inhibition by nicotinamide, blocking phosphorolytic cleavage of NAD+ and NADP+, (21,221 ergothioneine may regulate the conversion of glucose-6-phosphate to 6-phosphoglucona lactone by controlling the NAD+ required for the pyruvate to lactate conversion efEected by lactate dehydrogenase in anaerobic gLycoLysis. PlatoLets have an active pentose phosphate pathway whic:h i.s the chief source of CO2 produced from glucose. (23) They are also responsible for 25-308 of the total Lipid synthesis per unit of whole blood. (24) It would therefore appear that synthesis of acetyL CoA Erom pyruvate oxidized via the Krebs cycle must be important. Roth carnitine and eriJothioneine added in vitro to human platelets increase the i,roduction of CO2 from pyruvate, acetate, or 3-hydroxybutyrate. (25) For both compounds this occurs withouL_ increase in oxygen consumption indicating the effect ir, r!-lated to fatty acid metabolism and not respiration. T,iver, kidney, and red blood ceLLs aLl of which have significant pentose phosphate pathways also have been found tl, contain the highest amounts of ergothioneine. Frgothioneine may al50 have d respiratory effect in !;:l)'mC? in,;tances as it doubles the survival time of gllines pig ::;)ermatozoaMhile accelerating their rate of respiration with fructose a:; substrate and relieving the respiratory inhibition produced by P-chloro,ner._uribenzoat~. (261 If ergothioneine turns on a pentose phosphate pathway to sllpply ATP by anaerobic ylycolysis, a cell which contains 00 respiratory chain (erythrocyt_e) or a cel.1 which has had 355
no respiratory chain poisoned by cyanide, nitrite, or sulphydryl inhibitors might be expected to better survive the energy demand if ergothioneine content is augmented. Supporting such a function for ergothioneine are the observations that ergothioneine is antidotal to cyanide administration in the rat (27); protects from inhibition by sulphyaryl inhibitors the motility and aerobic and anaerobic fructolysis of spermatozoa (26,28); relieves the inhibition of anaerobic, but not aerobic, fructolysis in spermatozoa which is caused by Cu++ or H202 (28); and relieves the inhibition of lactate formation by sulphydryl inhibitors in Meyerhof extracts of rabbit skeletal muscle and alcoholic baker's yeast fermentations. (28) In red blood cells and liver cells, glutathione must be kept in the reduced form, a process requiring NADPH generated from the pentose phosphate pathway. The reduced glutathione is needed to convert methemoglobin, the Fe+++ nonoxygen binding form of hemoglobin, into Fe++ hemoglobin, the oxygen binding form. A cell or tissue in which ample ergothioneine insures an active pentose phosphate pathway would be better able to withstand stresses directed toward converting Fe++ to Fe+++ or not converting Fe+++ to Fe++. Nitrite ion causes such a stress and the resultant methemoglobinemia produced by ingestion of nitrate-contaminated well water is seen only in very young infants. (29) This may be because in both humans (30) and rats (31) blood ergothioneine levels are found to be lower in infants than in adults. (32) It is of more than passing interest to note the existence of another quaternary ammonium derivative of histidine. Post-translational modification of a histidine residue in the primary sequence of eukaryotic elongation factor-2 results in formation of diphthamide. When this residue is exposed to diptheria toxin, one of the imidazoyl nitrogens is ADP-ribosylated. (33,34,35) Ert may be open to similar ribosylation, either through an imidazoyl nitrogen or through a thioribosyl linkage at position 2 of the imidazoyl ring. Since Ert has been found in protein-bound form and since similar thiourylenes (the antithyroid methimazole and the antineoplastic 6-mercaptopurine) can be covalently attached to proteins (36,371, one can speculate that the expression of the physiological function of ergothioneine may be inhibited by diptheria toxin through ADP-ribosylation. Is erqothioneine
involved in cation transport?
Sulfur atoms resemble oxygen atoms when viewed using the Periodic Law. Oxygen has a 1~2 2s2 2~4 electronic configuration while sulfur has a 1~2 2~2 2~6 3~4
356
Since p-ofbitals require 6 electrons to be configuration. filled, both oxygen and sulfur can participate in two Because of its greater number of inner covalent bonds. shell electrons, sulfur is considerably less electronegative than oxygen and tends to form two single bonds where its oxygen analogues would form one double bond. In ergothioneine, the sulfur attached to the imidazole ring should be capable of binding and transporting monovalent cations such a H+, Na+, and K+ in a 1 to 1 molar ratio or divalent cations such as Cu++, Zn++, Ca++, or Mgtt using two ergothioneine per cation. Ergothioneine-monovalent cation complexes have not been investigated. Ergothioneine-divalent cation complexes have been formed, and 2 ergothioneine molecules are found to form extremely stable complexes with single atoms of Cu++ > Hq++> Zn++ > Cd++ > Co++ = Ni+t. (38, 39) Unfortunatley, Ca++-and Mg++ complexes were not studied. Since the administration of ergothioneine has been found to prevent cadium-induced teratogenesis in mice (40) it may be that poisonings caused by this and other metals may result from blocking the normal physiological function of endogenous ergothioneine. Epand (41) suggested that dietary ergothioneine may The play a role in the development of diabetes mellitus. reasoning is that ergothioneine has been found to be elevated in the blood of some diabetics. (42) Since ergothioneine levels have been found to have a strong affinity for Zn++, 138,391 and since Zn++ is important Ear the storage and secretion of insulin and glucagon from pancreatic islet cells, (43) perhaps this elevated ergothioneine is removing needed Zn++ and with it normal pancreatic reponse to glucose. Alternatively, abnormal diabetic pancreatic secretory activity may give rise to elevated blood ergothioneine levels. Interestingly, the removal of the rat pancreas produces a marked decrease in red blood cell ergothioneine. (44) Certain divalent metal-ion chelators are known to be diabetogenic compounds. Among these are certain analogues of 8-aminoquinoline, (43) the diabetogenic action paralleling the strength of the quinoline-Zn++ complex. Red blood cells deficient in glucose-6-phosphate dehydrogenase, an important pentose phosphate pathway enzyme, are more susceptible to the toxic action of certain 8-amino-quinolines. The question arises, does the physiological function of ergothioneine require Zn++ for expression? Like cancer, diabetes should be thought of as a malfunctioning process composed of many interrelating actions and reactions, any one of which may malfunction to produce the diabetic state. 357
Ergothioneine may be involved in Zn++ transport into cells. As an example, the red blood cell appears resistant to exchange with exogenous ergothioneine leading to the speculation that ergothioneine incorporation occurs only during erythropoesis. (1) Before maturation of red blood cells, at which time protein synthesis is terminated, large amounts (1 mg/ml packed cells) of the Zn++ metalloenzyme Does ergothioneine carbonic anhydrase must be synthesized. facilitate the Zn++ acquisition process? Does ergothioneine serve as a mediator for carbon dioxide fixation by carbonic Might similar ergothioneine Zn++-enzyme anhydrase? situations occurs in other cells? The reverse action of ergothioneine, the removal of Zn++ from Zn++ metalloenzymes, cannot be a general phenomena since the Zn++ metalloenzymes alcohol dehydrogenase, glutamate dehydrogenase and alkaline phosphatase are not inhibited by the addition of ergothioneine. (38) The capacity to take up, store, and decarboxylate amines may be quite widespread among polypeptide-secreting endocrine cells. (45) Perhaps ergothioneine plays a role in facilitating CO2 transfer in decarhoxylation reactions. Ergothioneine may be a cofactor decarboxylation reactions.
in carboxylation
or
The nitrogen atoms of a 2-thioimidazole are more It is nucleophilic than on the analogous 2-oxoimidazole. already known that the cofactor biotin, which contains an oxygen bearing ring, is involved in carbon dioxide Eixstion reactions, such as those catalyzed by the carboxylases for pyruvate, acetyl CoA, and propionyl Coil as well as Eor An immediate is methylmalonyl transcarboxylase. biotin-bound carbon dioxide (Fig. 5). One mig!lt suspect that ergothioneine would be even better at such a function. There is no experimental evidence to date which would indicate such a function, however, certain ergothioneine-like compounds with blocked nitrogens have been demonstrated to be antithyroids which may interEere with normal CO2 "transport" by ergothioneine.
358
“co;’ z ATP
r--
!
,c-o“co, E
r
_
t
----
CH~CH PROTEiN I +NlMel, ___-
2
;
ATP?
SH
I
,________-_-1
I _________---A
ERGOTHIONEINE
Figure 5. The thioimidazole bind carbon dioxide in carboxylation reactions in a manner similar to Thyroid or antithyroid ergothioneine.
nitrogens of ergothioneine may carbon dioxide transport, or decarboxylation reactions, that observed for biotin.
drugs may act on a process
involving
An antithyroid will decrease the basal metabolic rate This is measured directly by heat output or of an organism. indirectly by oxygen consumption or CO2 elimination. Various antithyroid drugs (Fig. 6) have a thiocarbamide structure suggesting a possible interference in an In addition, carnitine, ergothioneine mediated pathway. already seen to have certain ergothioneine-like molecular similarity, is an antithyroid. Initial experiments in which 50g female rats were given 2 mg/kg/day ergothioneine by injection over ten days followed by removal of their thyroids and assay of. iodincontent indicated ergothioneine and 2-thiouracil-treated rat thyroids had about 10% of the iodine contained in control thyroids. (.46) Subsequent studies in animals including humans failed to reproduce this initial observation. (47-311
359
r-f
II
I
Nwe
Nv
CH,CH$H,
CH,CH&H, I,,,,,-,-------
SH
Figure 6. There is an area of structural homology between ergothioneine and certain antithyroid drugs including A methimazole l3 propylthioimidazole (3 carbimazole D propylthiouracil
It would seem informative to check Nl-methyl, Nl-carbethoxy, or the g isomer of ergothioneine for thyroid or antithyroid activity. Additionally, if ergothioneine plays a role in both thyroid-antithyroid function and the diabetic condition, one might expect diabetics to have altered basal metabolism. Many diabetics are obese. Antithyroids are known to cause agranulocytosis. If these drugs are antiergothioneinics, granulocytes might be expected to contain ergothioneine or metabolites of ergothioneine. IE ergothioneine is incorporated into white blood cells only during early differentiation stages, one would expect to find ergothioneine in the bone marrow. This has been demonstrated. (52)
360
Ergothioneine or its derivatives, antihistaminic.
may be histaminic
or
Histidine is Ergothioneine is a histidine derivative. not pharmacologically active, but can be made so by histidine decarboxylase which liberates carbon dioxide and histamine, the latter being a very important and versatile molecule. Might ergothioneine suffer a similar fate, notably decarboxylation to a vasoactive amine (Fig. 7)3
/COO
l-l
CH,CH \ @NH,
/COO
\ V
N
0
HDC e
l-f
CH,CH, \ ,+NH,
G-3
CH,CH \ BN(Me),
CH,CH, \ ,,#(Me),
EDC?
NH
ERGOTHIONEINE
r-f
CH&$i aN(Me),
z
HERCYNINE
B
F'igur3 7. Two possible fates of ergothioneine metabolism. 4 - 'The formation of trimethyl thiohistamine. 3 - The Formation of trimethylhistaminr?. 361
Hercynine could be generated by oxidative desulfation. Subsequent decarboxylation would yield K-N-trimethylhistamine. Alternatively, decarboxylation of ergothioneine could Eorm Z-thio-Cd-N-trimethyl) histamine, which may OK may not be further metabolized. Hercynine has been found in blood (13.). Trimethyl histamine or trimethyl thiohistamine have not been knowingly assayed but may have been measured unknowingly in certain assays for histamine or ergothioneine. Similar compounds have been tested for pharmacological activity (Table 1). (53) Observe that&-N-methyl and "-N-dimethyl histamine have two to three times the gastric acid secretion activity as the same amount of histamine. Also observe that 2-thio-histamine has virtually no histaminic activity on smooth muscle Hl receptors. Methylating the imidazoyl nitrogens of histamine inactivates both HI and gastric acid secretion activites. Synthesis and testing of the compounds indicated should prove exceedingly informative.
R’
R
R”
R//f
Hl
H2
I --
H
H
1.0
1.0
HzCH3
--
H
H
1.0
2.5-3.0
WCH312
--
H
H
0.8
2-3
-_
H
0.006
H3
.R
CH.jZH,N
i-f R’
.N
vNxR” ’ ,,I R
/ Compounds of interest not tested
i
H3
CH3
H3
--
CH3
H3
--
H
SH
H(CH3)3
--
H
H
H(CH3j3
--
H
SH
--
H
SH
H3
TABLE 1
362
H
0
0 0
0.0005
? 0.0005
7
An example: Suppose a Interesting hypotheses emerge. cell carrying ergothioneine (eg-eosinophill) arrives at an inflammation site with the intent of controlling an inflammatory reaction through an antihistaminic pathway. Ergothioneine could be decarboxylated and the resultant molecule donates a methyl group to the imidazoyl nitroqen of any available histamine or vasoactive amine resulting in the Further suppose formation of two inactive compounds. Enter superoxide anion or modulation is not yet desirable. myeloperioxidase resulting in oxidative desulfuration of any histamines which thioimidazole, formingd-N-methylated Consider what may happen augment the histaminic response. under abnormal conditions. Trial and error experiments for antihistaminic compounds resulted in the development of Burimamide, Metiamide, and Cimetidine as specific H2 antogonists (Fig. 8). A thiocarbamide-like structure is found in each of these molecules and one wonders if these areas interfere with normal ergothioneine or egothioneine-like functions. One also wonders if closing the ring on these compounds (Fig. 8B) would give rise to more effective (specific and potent) H2 antagonists.
yc
CH,S II
kH2
l-f
I
CH,
I
“Wb.,,,,,, “V”” I HNCN
HNCN
I I
A
Figure 8.
6
A-Known H2 antagonists B-Potential H2 antagonists
A known cholinergic ergothioneine.
has structural
not yet tested
homolgy with
Pilocarpine, a cholinergic, has structural homology with ergothioneine (Fig. 9). Ergothioneine also has The obvious implication structural homology with choline.
363
is that ergothioneine, or a derivative of ergothioneine, might have cholinergic or anticholinergic action. Alternatively, ergothioneine or some derivative of ergothioneine may have an important function similar to but independent of the choline-acetylcholine system.
H
r-----
’
Me
/MeN” Me+ 8 (
01 6~ 1t-h
L____-_+----_
H CHOLINE
r_--_---__4
I
01
Me
61
/ MeN”
, Me+ i______+____,
eo
FH? - tRGoT”IONtlNtNtl=l ti
v\
/
/I hH
SH
Figure 9. Ergothioneine bears areas of structural homolgoy with choline/acetylcholine and the cholinergic drug pilocarpine. Ergothioneine has been reported to be exictatory on the electrical activity of the cerebellum of the decerebrate rabbit. (54) Ergohtioneine can also have excitatory effects on neurons in the brainstem. (55) Other reports dispute these findings. (56,571 CONCLUSIONS Presented are some easily testable hypotheses on possible physiological Eunctions for ergothioneine. It is important to keep in mind that ergothioneine itself may be inactive ant that it may be converted into an active form(s).
364
There exists a tantalizing possibility that ergothioneine may have different and even opposite physiological functions dependent on the route of its metabolism. It seems likely that within the framework of the hypotheses presented, a physiological function Ear ergothioneine will emerge.
365
REFERENCES 1.
Melville
DB.
2.
Hunter G. A new test for ergothioneine upon which is based a method for its estimation in simple solution and in blood filtrates. Biochem J, 1928;22:4.
3.
Hunter G. Colour reactions of thioglyoxalines (thioimidazoles) with sodium diazobenzene-p-sulphonate. J Chem Sot, 1930;2343.
4.
Jocelyn PC. The distribution of ergothioneine in blood as determined by a new method of estimation. Biochem J, 8;70:656.
5.
Hunter G. The determination of ergothioneine in simple solution and in blood. Can J Res, 1949;27:230.
6.
Fraser R. Blood ergothioneine levels in diabetes mellitus. J Lab Clin Med, 1950;35:960.
7.
Fraser R. Blood ergothioneine Clin Med, 1951;37:199.
8.
Smith EL, Tuller EF. The paper chromatographic detection of the free sulfur-containing amino acids and small peptides in whole blood and serum. Arch Biochem Biophys, 1954;54:114.
9.
Mann T, Leone E, Polge C. The composition of the stallions semen. J Endocrinol, 1956;13:279.
Ergothioneine.
Vitam Horm, 1959;17:155.
levels in diease.
J Lab
10.
Heath H, Rimington C. Further studies on seminal ergothioneine of the pig. Biochem J, 1957;65:369.
11.
Ackerman D, List PH, Menssen HG. Uber das vorkommen von herzynin neben ergothioneine in der samen-flussigkeit des ebers sowie in rinder erythrocyten und die biologische beziehung der beiden basen zueinander. 2 Physiol Chem, Hoppe Seyler's,1959;314:33.
12.
Carlsson J, Kierstan MP, Brocklehurst K. Reactions 1-ergothioneine and some other aminothiones with 2,2'-and 4,4 '-dipyridyl disulphides and of 1-ergothioneine with iodoacetamide. Biochem J,1974;139:221. 366
of
13.
Carlsson J, Kierstan MP, Brocklehurst K. A convenient spectrophotometric assay for the determination of l-ergothioneine in blood. Biochem J, 1974;139:237.
14.
Melville DB, Lubschez R. A method for the determination of ergothioneine in blood. J Biol Chem, 1953;200:275.
15.
Heath H, Toennies G. The preparation and properties ergothioneine disulphide. Biochem J, 1958;68:204.
16.
Mayumi T, Kawano H, Sakamoto Y, Suehisa E, Kawia Y, Hama 'I'. Studies on ergothioneine. V. Determination by high performance liquid chromatography and application to metabolic research. Chem Pharm BuLl, 1978;26:3772.
17.
Kaneko I, Takeuchi Y, Yamaoko Y, Tanaka Y, Fukuda T, Fukumori Y, Mayumi T, Hama T. Quantitative determination of ergothioneine in plasma and tissues by tic-densitometry. Chem Pharm Bull, 1980;28:3093.
18.
Fritz IB. Carnitine and its role in fatty acid metabolism. Adv Lipid Res, 1963;1:285.
19.
of
Kawano H, Otani M, Takeyama K, Kawai Y, Mayumi T, Hama Studies on ergothioneine. VI. Distribution and fluctuations of ergothioneine in rats. Chem Pharm BuLL, 1982;30:1760.
T.
20.
Yawano H, Higuchi F, Mayumi T, Hama T. Studies on s?rgothioneine. VII. Some eEfects of orgothioneine on gL:ycolytic metabolism in red blood celLs from rats. IChernPharm Bull, L982;30:2611.
21.
Grossman L, Kaplan NO. Nicotinamide riboside phosphorylase from human erythrocytes. I. ?hosFhor,Jlytic activity. J BioL Chem, 1958;231:717.
22.
Grossman L, Kaplan NO. Nicotinamide riboaide phosph0ryLas.e Erom human erythrocytes. II. Uicotinamide sensitivity. J Biol Chem,1958;23:727.
23.
Yerby GP, Taylor SM. The effect of added ATP on the :lathways of glucose utiLization by human washed ?L.atf+Letsin vitro. &J Lab CLin Med, 1967;69:194.
24.
tiar'isPA, Gelliorn A, Kidson C. Lipid synthesis in J Biol hAnan leukocytes, platelets, and urvthrocytes. [Chem, 1369;235:2573.
367
25.
Kerby GP, Taylor SM. Effect of carnitine and ergothioneine on human platelet metabolism. Proc Sot Exper Biol Med, 1969;132:435.
26.
Hama T, Okumura H, Tamaki N, Konishi T. Studies on ergothioneine. IV. Effect of ergothioneine upon spermatozoa from guinea pigs. Yakugaki Zasshi,1973;93:369.
27.
Trabucchi E. Richerche farmacologiche sulla ergothioneine. Boll Sot Ital Biol Sper, 1936;11:117.
28.
Mann T, Leone E. Studies on the metabolism of semen. VIII. ergothioneine as a normal constituent of boar seminal plasma. Purification and crystallization. Site of formation and function. Biochem J, 1953;53:140.
29.
Comly HH. Cyanosis in infants caused by nitrates well water. JAMA, 1945;129:112.
30.
Touster 0, Yarbro MC. The ergothioneine content of human erythrocytes: the effect of age, race, malignancy, and pregnancy. J Lab Clin Med, 1952;39:720.
31.
Mackenzie JB, Mackensie CG. The effects of age, sex, and androgen on blood ergothioneine. J Biol Chem,1957;225:651.
32.
Spicer SS, Wooley JG, Kessler V. Ergothioneine depletion in rabbit erythrocytes and its effect on Proc Sot Exper methemoglobin formation and reversion. Biol Med, 1951;77:418.
33.
Van Ness BG, Howard JB, Bodley JW. ADP-ribosylation elongation factor 2 by diptheria toxin. J Biol Chem,1980;255:10710.
of
34.
Van Ness BG, Howard JB, Bodley JW. ADP-ribosylation elongation Eactor 2 by diptheria toxin. J Biol Chem,1980;255:10717.
of
35.
Bodley JW, Van Ness BG, Howard JB. In: Smolson M, Sugimura T, eds. Novel ADP-ribosylations of regulatory enzymes and proteins. New York: Elsevier-North Holland, 1980;413-423.
368
in
36.
Hyslop RM, Jardine I. Metabolism of 6-thiopurines. Covalent binding of a 6-thiopurine metabolite to mammalian tissue protein in vivo. J Pharmacol Exp Ther,1981;218:629.
II.
37.
Poulsen LL, Hyslop RM, Ziegler DM. S-oxygenation of thioureylenes catalyzed by a microsomal flavoprotein mixed-function oxidase. Biochem Pharmaco1,1974;23:3431.
38.
Interaction Hanlon, DP. ions and metalloenzymes.
39.
Motohashi N, Mori I, Sugiura Y, Tanaka H. Metal complexes of ergothioneine.Chem Pharm Bull, 1974;22:654.
40.
Mayumi T, Okamoto K, Yoshida K, Kawai Y, Kawano H, Hama T, Tanaka K. Studies on ergothioneine. VIII. Preventive effects of ergothioneine .on cadmium-induced teratogenesis. Chem Pharm Bull, 1982;30:2141.
41.
Epand RM. The role of dietary ergothioneine development of diabetes mellitus. Med Hypotheses,1982;9:207.
42.
Fraser R. mellitus.
43.
Lazaris JA, Bavelskyi ZE. Primary insulin insufficiency after blocking of zinc in pancreatic beta-cells. Endocrinol Exp (Bratisl), 1981;15:99.
44.
Beatty CH. Levels of ergothioneine in the red blood cell in experimental diabetes. J Biol Chem,1952;199:553.
45.
Pearse AGE. Mem Sot Endocrinol,
46.
Lawson A, Rimington C. Antithyroid activity ergothioneine. Lancet, 1947;252:586.
47.
Searle CE, Lawson A, Hemmings AW. Antithyroid substances. I. The mercaptoglyoxalines. Biochem J,1950;47:77.
of ergothioneine with metal J Med Chem, 1971;14:1084.
in the
Blood ergothioneine levels in diabetes J Lab Clin Med, 1950;35:960.
369
1971;19:543. of
48.
Heath H, Rimington C, Searle CE, Lawson A. Some effects of administering ergothioneine to rats. Biochem J, 1952;50:530.
49.
Baldridge RC. Blood ergothioneine Nutrition, 1955;56:107.
50.
Wilson ML, McGinty DA. Antithyroid activity ergothioneine. Am J Physiol, 1949;156:377.
51.
Astwood EB, Stanley MM. Antithyroid activity ergothioneine. Lancet, 1947;252:586.
52.
Lutwak-Mann C. in bone marrow.
53.
Histamines and anti-histamines. Part II. M Kocha e Silva, ed. Berlin: Springer-Verlag, 1966;182:427.
54.
Crossland J, Mitchell JF, WoodrufE GN. The presence ot ergothioneine in the central nervous system and its probable identity with the cerebellar factor. ,J Physiol, 1966;182:427.
55.
Avanzino GL, Bradley PB, Comis SD, WolstencroEt JH. A comparision of the actions oE ergothioneine and chlorpromazine applied to single neurons by two different methods. Int J Neuropharmacol, 1966;5:331.
56.
Krnjeric K, Randic M, Straughan DW. Ergothioneine central neurons. Nature, 1965;205:603.
57.
Briggs I. Ergothioneine in the central nervous systems. J Neurochem, 1972;19:27.
and dietary oats.
Some aspects of phosphorous Biochem J, 1952;52:356.
370
,I
of
of
metabolism
and