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The metabolism of methyl mercaptan in the intact animal
The Metabolism
of Methyl Mercaptan in the Intact Animal’
E. S. Canellakis2
and Harold
Tarver
From the Department of Physiological Chemistry, University California School of Medicine, Berkeley, California Received
September
of
24, 1952
In the previous communication it has been shown that methionine forms methyl mercaptan (methanethiol) when incubated in the presence of mitochondria from rat liver (1). The present investigation is concerned with some of the qualitative aspects of the metabolism of methyl mercaptan. To facilitate the investigation, the mercaptan labeled with both Cl4 and P6 has been used. The appearance of the labeled atoms in excretory channels and in various amino acids in the proteins of animals injected intraperitoneally with the mercaptan has been determined. In addition, a search for the Cl4 in creatine-creatinine and choline has been made. The results show that methyl mercaptan is rapidly oxidized and that the labeling of a variety of substancesis qualitatively the same following the administration of methyl mercaptan as after methionine-CY4H3. When methyl mercaptan-F5 was injected none of the label appeared in the liver protein methionine, indicating a lack of reversal of the desulfuration reaction. EXPERIMENTAL General Description of Experiments The of 04
first experiment from methyl
1 This investigation National Institutes 2 Present address: Madison, Wise.
was carried mercaptanCi4
out with the object of determining appearing in the respiratory
was supported (in part) by a research of Health, U. S. Public Health Service. Dept. of Physiological Chemistry, University
446
the amount gases as ungrant
from
of Wisconsin,
the
METHYL
MERCAPT.\N
MET.\BOLISM
447
changed mercaptan antI as carbon dioxide, the amount appearing in the urine, antI the relnt,ive activity of the tissues. The animal used, :t 180-g. male rat, was given :t dose of 0.3 mg. methyl mercnptan (1.03 X 106 counts/min.) intraprritonally. Immediately after injection t.he rat. was placed in an all-glass metabolic :Ipp:Lratus which allowed the collection of all the respirat,ory gas and the urine. Following :L 6.hr. collection period. the animal W:IS s:lcrificed :tncl that tissue pr” 1 t,ins were isol:~t,ed. The second experiment had :ts its main object the preparation of highly radio:lct.ive liver protein which would :~llow of the determimttion of the activity irl the individual amino acids, and t,he investigation of the r:tdioltctIivity of tissue> cholinr and crrat.ine. To this end a 200-g. malt rat was given an intr:l~)c,ritollc:tl dose of I .-I mg. of methyl mercnpt.an containing 2 X 1W counts/min. as (‘I’. The i~nd the animal \~:Ls s:tcrifi(u(x(l tlosr was given in nine aliquots at I-hr. inkrvals, 2 hr. :ift,er the last, doss. The liver prot,ein was isolakd and hydrolyzed. :md the amino acids were separated on Dow-es 50. Radiottctivity appeared as various peaks in the successive portions of t,he eluttnt, acid. The nature of the sulA:tn(*t’s in t,hr peaks was determined hy paper chrom:tt,ogrxph?. Choline an,1 crc:~l ilux \VPW isolated as derivatives from suitable estract.s of thp Gssurs. Thr third experiment was carried out with methyl mercaptnn-Pa5 in ortl~r to gain some information :LS to the extent of oxidation of the sulfur to sulf:ttc* :t~~tl in orrlrsr to SW if t,he r:rdio:tct,ive sulfur appeared in t hr methionine or ryst inc of t 1~8 liver protrin. For the purpose :t male rat of 2W g. bv:~s given 2 mg. of thr m(‘1’c:tpt:tn (1.!) X IO6 counts/min. of W) :ts a dose tiivitkd into four portions given at 2.hr. int.nrv:tls intrtLperitonenll\-. The Ltnim:tl w:ts sacrificptl 2 hr. after the Ias ~losc:, thrb urine being collected during the 8.hr. period of the experiment. 1,ivc.r I)rotrin was ~~re~xtrrtl and its cystine and mrthioninr werr isol:tkd after hytirol!.sis with hydroiodic arid in the form of the culn~,us salts of cystc,ino antI Ilonlo cyst,einc For comp:tr:ttive purposes :t fourth rsperimrllt was done w-it h t lrc r)i)j(.c.t 01 :~ntl of comparing the estcnt ot I:tl)rling the, proteins wit,h I.-mcthiouine-CL’H:I, I:t~)c~ling of the various amino acids which occurrrtl with thxt founcl after tllrl methyl merc:~ptan-C1+. In this experiment, a m:d(x mouse of 20 g. was injcctctl with :t total of 11 mg. of the amino acid, given in tx\-cj doses: the first of 4 mg. followrd 1,~ t,hr> second after 1 hr. (t,otal r:tdio:ictivity of 3 X 10’ c*ount~s/min. I. The animal was sacrificed 6 hr. after the first injection, livcxr proteins WPIY sc'lw r:tted . and the distribution of the radio:tctivit !- among the amino acids in the, Irytlrolyzat ~1 was drt,rrmincd t)>- thp same mrthotls usrct in the sworlcl c~xl)c~rilnc~nt
Methods Lnbclletl .Il~fhq( dlercuptnn. r:tptnn, the 1:theled starting r:tt,ories; for thr C’L4-l:tl)e1ed AI. Tolhert, md Miss Patricia these workers for supplying To 20 mg. of thiourea-W the I mmole W:LS dissolved in of methyl iollide in 1 ml. of
For the synthesis of t hr Ss5- I:thelrd methyl nior~ material wns thiourc:t ohtnined from Ahhott l:tl )ocompound, labeled met,tryl iodide prepared t)!. Dr. 1i. T. Adams was used. Our I)c.st th:tnks :~rc (IW 1 I) the int,ermediates. was added 56 mg of t ht, urll:~l~elml wrnpourltl. :rtltl 1 ml. of warm alcohol. To this was :d&xl 1.5 ~nmolw alcohol folloxr~l by 300 mg. of picric :r.citl. Thcl nli\-
4448
E.
S.
CANELLAKIS
.4ND
HAROLD
TARVER
ture was warmed in a closed apparatus to dissolve the picric acid and the product, S-methylisothiourea picrate crystallized out on standing. The yield was 266 mg. (83% theory) of a product with a melting point of 215” (uncorr.) [literature (2) 221”]. The Cl”-labeled material was made in a similar fashion except that a 10% excess of thiourea was employed rather than a 50% excess of methyl iodide. Labeled I,-Methionine. The preparation is described in the previous communication (1). Solutions of Mercaptan for Injection. Methyl mercaptan solutions for injection were prepared from aliquots of the S-methylisothiourea picrate by heating with an excess of 5 N sodium hydroxide. The mercaptan thus generated was cooled to 0” and transferred into 4 ml. of 0.1 N sodium hydroxide by flushing the apparatus with nitrogen. After removing a 0.02-ml. aliquot from the basic solution for the determination of radioactivity, the solutions were neutralized with ice-cold phosphoric acid in a hypodermic syringe and used immediately for injection. Radioactivity was determined in the case of methyl mercaptan-S35 by adding t.he aliquot to Pirie’s reagent (3), allowing it to stand at room temperature 3-4 hr., and then digesting in the usual fashion in the presence of carrier sulfate. The radioactivity in the sulfate was determined by counting benzidine sulfate with a Geiger-Miiller counter with an end-window tube. Radioactivities of the basic solutions of methyl mercaptan-Cl4 were determined by combustion of the aliquots in the presence of dextrose and precipitation of barium carbonate by the Van Slyke and Folch method (4-6). Separation of Methyl Mercaptan and Carbon Dioxide. Methyl mercaptan reacts with isatin in cont. sulfuric acid to give a green color (7), and it was found possible to use this reaction both to give an indication of the presence of the mercaptan and t,o trap it. When lo-20 mg. of methyl mercaptan containing 5 X lOa counts/min. of Cl’ was passed through two wash bottles containing the isat,in reagent (10 mg. isatin in 100 ml. cont. sulfuric acid), followed bJ two wash bottles containing sodium hydroxide, only the contents of the first wash bottle turned green. All the radioactivity was present in this bottle as determined by combustion (4-6) with none in either the second containing the isatin reagent or in the third and fourth containing the base. In the metabolic experiments, therefore, a similar train of wash bottles was used to collect any mercaptan excreted by the animals and to trap the respiratory carbon dioxide. Determinations on Urine. The Cl” in the urine was measured by burning an aliquot (4-6). The total 536 in urine was determined by oxidation of an aliquot with Pirie’s reagent with added sulfate as a carrier. Sulfate was precipitated and counted in the form of the salt with henzidine. Inorganic sulfate was determined by direct precipitation of an aliquot containing added sulfate, and total sulfate by precipitating after evaporating down in the presence of hydrochloric acid. Preparation of Tissue Proteins. To obtain protein samples for the separation of amino acids, tissues from the animals were homogenized and denatured with trichloroacetic acid (TCA). The TCA wash was repeated three times. Then they were washed several times with boiling alcohol, with hot alcohol-ether, and with ether. Separation of Amino Acids from Proteins. The dried protein samples were pulverized and lOO-mg. samples were hydrolyzed for 12-16 hr. at 120” with 200
times their weight of 6 IV hydrochloric acid. The hytlrol~~z:ttes wow t~v:rlwr:ltc~tl to dryness at reduced pressure and, aft,er taking up in water. the humin ww wntrifuged down and discarded. To remove thr dic:rrt)osylic wids from the tt!.clrolyznt,e, the solution was passed through a column of the txtsic resin, Amlwrlitc~ II<-113. A further separation was t,hrn made 1)~ passing the effluent Induced to :I small volume through a column of Dowes 50 of 2llGNo mesh in thr xcitl fomI :I( c9rtlitlg to 111~ method of Skin and AIoore (8). Samplw \vr’rr wllrcted in polo c~thylrnc~ planrhets using a fra~tioIl.collcctinF: :tpp:w:rtus of the I ylw tlrwrilwd t)y I,ic:n, I’r;trrson, and Greentwrg (9). For elution tlw follo~ving wcw usr~l ill t hc order given: wtcr, I .5 :md 2.5 S hydrochloric acids Iwth 0.3.5 satur:tte(l \vith sulfur dioxide nt room temper:lturP, :tnd 1 ,V antI 6 .V t1\~dro~hlori~* :tcills. The* sulfur dioxide was employed in :i lxtrtially successful at teml)t t 0 minimizct t hti fnrnxttion of met hionine sulfoxide. The acid was rcmnvtd from t hf s:ml~~lcs OII t hex pl:tnchets, :IIIII the radioactivities were drkrmined tl). counting using :t gastlow t utw. The n:tt,urr of the radioactive amino acids in thr various pwks \V:S determined Iry their position wid t)y running tno-tlimt~nsiori:tl chrnm:tt oprams oti :\liquots t:tk(ln from the peaks and comlxtring the travel of thf, rlinfl~driI~-l)osit ivc’ material and the radioactivity with st:rndards of :~pproprixtc amino xcitls. In addition. si~~~lr-tlime~lsiorl:L1 chromatogrnms of :tlicluots from alternate l)l:mch~~ts were m;& in orller to locate the nnnr:ttlio:~ctive xnino witIs. For c*hriml:ttog r:tl)hy. What man So. -I paper wws employed and either l)hr,rrol~-\\:ltcr, trr Ibut :11101 :twtir acicl mixtures. 13~ this means it w:ts found that thr amino witIs \VPI‘C c~lutwl from the> I)owex in the order given I)y Stein and iLlr)orr (N), witI1 tlw t~xwl)tiori that phrr~yl:d:mine appeared before rathrr than after arginine. fsolnlio/r oj’ (‘holine and C’reufine. Crr:ttinc \vas isolntc~tl in thr form of cr(y:It i tlinc pic.r.:ltcl from the aqueous cstracts of the tissues anal thr liwr i 101. Th~l l~ro(l~ urt was rwrystallized twice from the potassium picr:itc~ l)icric :wi(l rriistuw :trl~l its specific rndioactivit> drtermincd. Two mow rrc.r?st:llliz:ltiolls from thr same’ solvent \ver(’ then carried out :IS well :ts OIIP from w:ttcsr. .\ftctr r,:tc*h rwy!.st:1lliz:~ tinn thr spwitic :I.ctivity \v:ts rctletrrminrd. (‘holillc~ was isolated as thr chloropl;tt in:Lie from the :Llcotlnl :1ntl :LIw1IoI ether vstr:tc*ts of all the tissues coml)inc~d (11). After tlctt~rminstion of its slwcific* r:ldio:tctivity, :t part was degrxlrd to trimrthylxnille with :tlk:llinr~ ~wI~:III K:tn:ctp (12). The trimcthyl:tminr wxs also isol:lt<~(l as t ht> c.hlol,ol)l:rtitl:1t(. four t 111% det(~rmitiiLt ion of its CL4 content. Isolntir~rr 0.f Jlethionine nntl C’!ys!eine. In Fkpt :3 the sulfur-~l)llt:lirlirlg amino :wills wcr(. frwtiomttrd from aliquots of :t hydroiodic xcitl digest of 500 trig. of liver lnwtcin t)y the met,hod of Beach nnd Teque (13) in orclcar to avoid esccssivc~ losses t)y humin formation. From one aliquot thrl cuprous salt of cystcinr w:w prelwwd xnci from t,hr other the mixed salts of yysteinc x11(1 l~onioc~stc~it~r :w drscritwd t)>- thrsc xuthors. Ikr~rc~dation o,[ Alefhionine and Serinr. ilIet.hionint~ from thr al)l)rol)ri:lt(~ i’r:t(. tions from the column was degraded 1)~. digest,ion with h>-driodic xcid (11, 1.51 The mcltllyl iodide evolved was swept over into an :tlcoholic solnt ic)ll of t rimc~th~ I :Imine and isolated as t~etr:tmet~hylsmmonium iodide (161. Scrinc from the column was degraded with periwlic will. :III~ thcx formal111~ l~~.tl~ fornwl from 1he @-carbon was converted to the ~limwlon clcsriwt iw ( Ii I.
E. S. CANELLAKIS
450
AND HAROLD TARVER
RESULTS
Table I shows the amounts of Cl4 appearing in excretory channels following a single dose of methyl mercaptan to a rat. From the results it is evident that the methyl mercaptan in part escapesunchanged but that a large fraction of the dose is very rapidly oxidized to carbon dioxide. In the 6-hr. period of the experiment, about 40% of the dose appeared in the respiratory carbon dioxide. The results of Expt. 1 are substantiated by Expt. 3 in which mercaptan-P5 was employed. Again TABLE Distribution
I
of Excreted Radioactivity Following the Administration of Methyl Mercaptan-Cl* or P6
Expt. 1. Single dose of methyl mercaptan-Cl4 rat. Animal sacrificed after 6 hr. Expt. 3. Multiple (1.4 mg. total). Rat sacrificed after 8 hr.
Period
Expt.
1
(0.3 mg.) intraperitoneally to dose of methyl mercaptan-S36
Respiratory
carbon dioxide
hr.
%
o-l
29.2 6.2 3.8 1.6
l-2 2-4 4-6
Volatile
3
sulfur excreted
Fraction of dose excreted
O-l
6.4
l-2
0.0
Urine
O-6
2.3
Urine Total sulfur Total sulfate Inorganic sulfate
O-8 32.
31. 29.
a great oxidation is demonstrated. It might be anticipated that there should be some correspondence between the mercaptan oxidized to carbon dioxide and that appearing as sulfate in the urine. The observed discrepancy must be attributed to the fact that in one casethe mercaptan was given in a single dose whereas in the other the dose was divided, so that the last part was given only 2 hr. before sacrificing the animal. When the tissues were examined it was found that they retained considerable activity as shown by the specific activity figures given in Table II. It may be noted that in Expt. 2 the retention of the Cl4 by the proteins of the intestinal mucosa is’relatively lower than it is when
radioactive amino acids are administered to an animal (18). When the liver protein preparation obtained in Expt. 3 was washed with monot’hioet,hylene glycol (2-mercaptoethanol) about half of the activity was removed. Presumably this loss is due to the binding of methyl merc:aptalI as such to t’he protein rather t,han due t’o the introduction of a&vity from the protein into amino acid which in turn is iworporat’ed into the protein. Since the last dose of methyl mercaptan was gi\-en only 2 hr. before t’he animal was sacrificed, this observation is understandable 011 the basis of the results described in the previous communication (I). III Espt. 1 tbe label in the proteins from the animal was stable to monothioethylene glyc.01. Six hours after the administration, all t,he methyl mewnptnn may have heen oxidized. These results are similar t.o those
4
Live1 Kidney Spleen Lung Testis
17.5 11.1 9,s 11.5 8.5
Liver Liver”
10.1 4.9
‘I Liver protein preparation ous communicxbtion (l)].
Plasma protein Intestinal mucow Muscle Erythrocytes
washed with monothioet,hylene
22.7 16.7 2.2 0
glycol [see previ-
found by Lee and co-workers following the administrat8ion of cystine (19). When t(he wtivity of the cysteine and of the homocysteine-cystine from the liver protein (500 mg. with approx. 2500 counts/min. of 8”” stable to monothioethylene glycol) was examined, all samples had zero activity. It would appear, t’herefore, that the SS5associated with the protein in tbis experiment, was in the form of sulfate, and that the process lvhich leads to the formation of methyl mercaptan from methionine in rat, liver mitochondria is not reversible to any significant extent. The column radiograms of the hydrolyzates of the liver proteins of the rat, given methyl mercapt*a.n-P and of the mouse given L-methiol~i~w(“4H:j separated on I>o\res 50 are shown in Figs. I and 2, respeca-
452
E.
R.
CANELLAKIS
.4ND
H.4ROLD
TARVER
tively. It is evident from the first figure that Cl* from the mercaptan has been converted to serine (peak B) and methionine (peak h’), and from the second figure that the Cl4 of the methyl group of methionine has been converted to serine. In both experiments, a part of the methionine appeared as the sulfoxide (peak C), which occurred in approximately the same location as alanine. In both experiments, a remarkable amount of activity was present in the serine peak. In both radiograms 12,000 -
t I3
9,000 -
i ci cj 3,000 -
E A /\,L 100
k ti&---I.5
C
I 6 200 300 N. HCI t SO~----+2.5
Fraction
I 400 N. HCI HGI + SO,-
Number
FIG. 1. Column radiogram of the hydrolyzate of 1.36 g. of liver protein from the rat used in Expt. 2 after administration of methyl mercaptan-C”. Column: 113 X 1.4 cm. filled with Dowex 50, 2oOL4OO mesh in acid form. Solvents: water, 96 ml.; 1.5 N HCl with SO*, 470 ml.; 2.5 N HCI with SOZ, 420 ml.; 4.0 N HCl, 315 ml.; 6.0 N HCI, 220 ml. Location of peaks: serine, 204 ml. 1.5 N HCl with SO?; methionine, 260 ml. 2.5 N HCl with SO?.
there appeared small peaks, A and D, which could not be satisfactorily identified, although A may be due to aspartic acid. The similarity in these two radiograms is rather striking; they differ chiefly in that the administration of mercapt,an-C I4 leads to a protein richer in labeled serine and poorer in methionine than that obtained after methionineC14H3. Little activity was noted in the cystine peak, which may have been due to the loss of most of this amino acid in the form of humin.
METIIYL
MERC‘APT:iN
METABOLISM
-453
No significant radioactive peaks were found in those fractions not shown in the chromatograms. When the serine and methionine separated by the cbolumn radiography from the hydrolyzate of the liver protein of the rat given methyl mercqt,an-Cl-’ (Expt. 2) were suhjec+ed to degradation it was found, as shown it1 Table III, fhaLt bot,h the methyl group of methionine and t’hc 15,000 t
b+ I-$0+
B
loo 200 1.5N. HCI t So, --+
Fraction
300 2.5 N. HCI + SC+-
Number
IJrc;. 2. (‘olunm radiogram of the hydrolq-zate of 0.16 g, of liver protein from tlui mouse used in I
@c*arbon of serine were highly active. The results also show t,he high wti\‘ity of the choline isolated from the carcass of the same animal. Moreover, the activity of the trimethylamine accounts for most of that present’ in the choline. There may be some activit,y in the ethauolamine portion of the molecule due to decarboxylation of serine, which has becwme labeled in the p-carbon. Creatinine from t,he body creatine was also c1uit.e a&ve.
354
E.
S.
CANELLAKIS
AND
HAROLD
TARVER
It is obvious from these experiments that the metabolism of the methyl group of methyl mercapt’an is in some respects similar to that of the methyl group of methyl alcohol as shown by a comparison of the results reported here with those obtained by V. du Vigneaud and coworkers (20, 21), and that the results are explicable on the same basis, namely oxidation of the methyl group t’o an intermediate which may either appear as the P-carbon of serine or may be reduced again to reappear as a labile methyl group. TABLE Distribution
III
of Activity
Ezpt. 2. Multiple after 10 hr.
in Vutious Constituents of the Rat Administration oj Methyl Mercaptan-Cl4 dose of methyl
mercaptan-Cl4
(1.9 mg.
total).
Following Rat
the sacrificed Specific activitya c0unfslmin.l +w.
Methyl
group of methionine from liver protein isolated as tetramethylammoniumiodide................................................. &Carbon of serine from liver protein isolated as dimedon. Creatine isolated from tissues as creatinine picrate. Recrystalliaationsh l.......................:............................ 2 .._..._...__.................. .._... .._........ 3.. Choline isolated from tissues as chloroplatinate. Trimethylamine chloroplatinate from degradation of choline above.
24.7 108.5 20.3 20.3 20.5 179. 187.
a Values given are not comparable because they are not on a molar basis and are not corrected for self-absorption. Corrected activities would give no additional information because of the large dilutions with the preformed compounds largely unknown as to amount and turnover rate. b See under Methods for details. SUMMARY
In experiments in which methyl mercaptan-CL4 or S35had been administered to rats in vitro the following has been found: 1. Both the carbon and the sulfur of the mercaptan are rapidly oxidized to carbon dioxide and sulfate, respectively. 2. The methyl carbon of the mercaptan is converted to the p-carbon of serine and methyl groups of methionine, choline, and creatine. 3. Sulfur from methyl mercaptan does not appear to any significant extent in the methionine or cystine of liver protein.
METHYL
MERCAPTAN
435
METABOLISM
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ix:~ ( I !)-I!) 1 I?. T,EVISE, 11.. .ASI) TARVER. II., ./. Rid. (‘hem. 164, $27 (1950). IS. TARVXR. 11.. in (;REENBF,RG, D. hf., The Amino Acids and I’rokins, Thonxts Puhl. Co., Springfield, 1951. I!). LEE, ?r;. 11.. ANDERSON. J. T., ~ZILLER, 11.. ASI) Wrr.r.r.\ar~. I{., J. Viol. 192, 733 (1951). 20.
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(‘. f'hew
(1950). R..
REX-
(’
of Methyl Mercaptan in the Intact Animal’
E. S. Canellakis2
and Harold
Tarver
From the Department of Physiological Chemistry, University California School of Medicine, Berkeley, California Received
September
of
24, 1952
In the previous communication it has been shown that methionine forms methyl mercaptan (methanethiol) when incubated in the presence of mitochondria from rat liver (1). The present investigation is concerned with some of the qualitative aspects of the metabolism of methyl mercaptan. To facilitate the investigation, the mercaptan labeled with both Cl4 and P6 has been used. The appearance of the labeled atoms in excretory channels and in various amino acids in the proteins of animals injected intraperitoneally with the mercaptan has been determined. In addition, a search for the Cl4 in creatine-creatinine and choline has been made. The results show that methyl mercaptan is rapidly oxidized and that the labeling of a variety of substancesis qualitatively the same following the administration of methyl mercaptan as after methionine-CY4H3. When methyl mercaptan-F5 was injected none of the label appeared in the liver protein methionine, indicating a lack of reversal of the desulfuration reaction. EXPERIMENTAL General Description of Experiments The of 04
first experiment from methyl
1 This investigation National Institutes 2 Present address: Madison, Wise.
was carried mercaptanCi4
out with the object of determining appearing in the respiratory
was supported (in part) by a research of Health, U. S. Public Health Service. Dept. of Physiological Chemistry, University
446
the amount gases as ungrant
from
of Wisconsin,
the
METHYL
MERCAPT.\N
MET.\BOLISM
447
changed mercaptan antI as carbon dioxide, the amount appearing in the urine, antI the relnt,ive activity of the tissues. The animal used, :t 180-g. male rat, was given :t dose of 0.3 mg. methyl mercnptan (1.03 X 106 counts/min.) intraprritonally. Immediately after injection t.he rat. was placed in an all-glass metabolic :Ipp:Lratus which allowed the collection of all the respirat,ory gas and the urine. Following :L 6.hr. collection period. the animal W:IS s:lcrificed :tncl that tissue pr” 1 t,ins were isol:~t,ed. The second experiment had :ts its main object the preparation of highly radio:lct.ive liver protein which would :~llow of the determimttion of the activity irl the individual amino acids, and t,he investigation of the r:tdioltctIivity of tissue> cholinr and crrat.ine. To this end a 200-g. malt rat was given an intr:l~)c,ritollc:tl dose of I .-I mg. of methyl mercnpt.an containing 2 X 1W counts/min. as (‘I’. The i~nd the animal \~:Ls s:tcrifi(u(x(l tlosr was given in nine aliquots at I-hr. inkrvals, 2 hr. :ift,er the last, doss. The liver prot,ein was isolakd and hydrolyzed. :md the amino acids were separated on Dow-es 50. Radiottctivity appeared as various peaks in the successive portions of t,he eluttnt, acid. The nature of the sulA:tn(*t’s in t,hr peaks was determined hy paper chrom:tt,ogrxph?. Choline an,1 crc:~l ilux \VPW isolated as derivatives from suitable estract.s of thp Gssurs. Thr third experiment was carried out with methyl mercaptnn-Pa5 in ortl~r to gain some information :LS to the extent of oxidation of the sulfur to sulf:ttc* :t~~tl in orrlrsr to SW if t,he r:rdio:tct,ive sulfur appeared in t hr methionine or ryst inc of t 1~8 liver protrin. For the purpose :t male rat of 2W g. bv:~s given 2 mg. of thr m(‘1’c:tpt:tn (1.!) X IO6 counts/min. of W) :ts a dose tiivitkd into four portions given at 2.hr. int.nrv:tls intrtLperitonenll\-. The Ltnim:tl w:ts sacrificptl 2 hr. after the Ias ~losc:, thrb urine being collected during the 8.hr. period of the experiment. 1,ivc.r I)rotrin was ~~re~xtrrtl and its cystine and mrthioninr werr isol:tkd after hytirol!.sis with hydroiodic arid in the form of the culn~,us salts of cystc,ino antI Ilonlo cyst,einc For comp:tr:ttive purposes :t fourth rsperimrllt was done w-it h t lrc r)i)j(.c.t 01 :~ntl of comparing the estcnt ot I:tl)rling the, proteins wit,h I.-mcthiouine-CL’H:I, I:t~)c~ling of the various amino acids which occurrrtl with thxt founcl after tllrl methyl merc:~ptan-C1+. In this experiment, a m:d(x mouse of 20 g. was injcctctl with :t total of 11 mg. of the amino acid, given in tx\-cj doses: the first of 4 mg. followrd 1,~ t,hr> second after 1 hr. (t,otal r:tdio:ictivity of 3 X 10’ c*ount~s/min. I. The animal was sacrificed 6 hr. after the first injection, livcxr proteins WPIY sc'lw r:tted . and the distribution of the radio:tctivit !- among the amino acids in the, Irytlrolyzat ~1 was drt,rrmincd t)>- thp same mrthotls usrct in the sworlcl c~xl)c~rilnc~nt
Methods Lnbclletl .Il~fhq( dlercuptnn. r:tptnn, the 1:theled starting r:tt,ories; for thr C’L4-l:tl)e1ed AI. Tolhert, md Miss Patricia these workers for supplying To 20 mg. of thiourea-W the I mmole W:LS dissolved in of methyl iollide in 1 ml. of
For the synthesis of t hr Ss5- I:thelrd methyl nior~ material wns thiourc:t ohtnined from Ahhott l:tl )ocompound, labeled met,tryl iodide prepared t)!. Dr. 1i. T. Adams was used. Our I)c.st th:tnks :~rc (IW 1 I) the int,ermediates. was added 56 mg of t ht, urll:~l~elml wrnpourltl. :rtltl 1 ml. of warm alcohol. To this was :d&xl 1.5 ~nmolw alcohol folloxr~l by 300 mg. of picric :r.citl. Thcl nli\-
4448
E.
S.
CANELLAKIS
.4ND
HAROLD
TARVER
ture was warmed in a closed apparatus to dissolve the picric acid and the product, S-methylisothiourea picrate crystallized out on standing. The yield was 266 mg. (83% theory) of a product with a melting point of 215” (uncorr.) [literature (2) 221”]. The Cl”-labeled material was made in a similar fashion except that a 10% excess of thiourea was employed rather than a 50% excess of methyl iodide. Labeled I,-Methionine. The preparation is described in the previous communication (1). Solutions of Mercaptan for Injection. Methyl mercaptan solutions for injection were prepared from aliquots of the S-methylisothiourea picrate by heating with an excess of 5 N sodium hydroxide. The mercaptan thus generated was cooled to 0” and transferred into 4 ml. of 0.1 N sodium hydroxide by flushing the apparatus with nitrogen. After removing a 0.02-ml. aliquot from the basic solution for the determination of radioactivity, the solutions were neutralized with ice-cold phosphoric acid in a hypodermic syringe and used immediately for injection. Radioactivity was determined in the case of methyl mercaptan-S35 by adding t.he aliquot to Pirie’s reagent (3), allowing it to stand at room temperature 3-4 hr., and then digesting in the usual fashion in the presence of carrier sulfate. The radioactivity in the sulfate was determined by counting benzidine sulfate with a Geiger-Miiller counter with an end-window tube. Radioactivities of the basic solutions of methyl mercaptan-Cl4 were determined by combustion of the aliquots in the presence of dextrose and precipitation of barium carbonate by the Van Slyke and Folch method (4-6). Separation of Methyl Mercaptan and Carbon Dioxide. Methyl mercaptan reacts with isatin in cont. sulfuric acid to give a green color (7), and it was found possible to use this reaction both to give an indication of the presence of the mercaptan and t,o trap it. When lo-20 mg. of methyl mercaptan containing 5 X lOa counts/min. of Cl’ was passed through two wash bottles containing the isat,in reagent (10 mg. isatin in 100 ml. cont. sulfuric acid), followed bJ two wash bottles containing sodium hydroxide, only the contents of the first wash bottle turned green. All the radioactivity was present in this bottle as determined by combustion (4-6) with none in either the second containing the isatin reagent or in the third and fourth containing the base. In the metabolic experiments, therefore, a similar train of wash bottles was used to collect any mercaptan excreted by the animals and to trap the respiratory carbon dioxide. Determinations on Urine. The Cl” in the urine was measured by burning an aliquot (4-6). The total 536 in urine was determined by oxidation of an aliquot with Pirie’s reagent with added sulfate as a carrier. Sulfate was precipitated and counted in the form of the salt with henzidine. Inorganic sulfate was determined by direct precipitation of an aliquot containing added sulfate, and total sulfate by precipitating after evaporating down in the presence of hydrochloric acid. Preparation of Tissue Proteins. To obtain protein samples for the separation of amino acids, tissues from the animals were homogenized and denatured with trichloroacetic acid (TCA). The TCA wash was repeated three times. Then they were washed several times with boiling alcohol, with hot alcohol-ether, and with ether. Separation of Amino Acids from Proteins. The dried protein samples were pulverized and lOO-mg. samples were hydrolyzed for 12-16 hr. at 120” with 200
times their weight of 6 IV hydrochloric acid. The hytlrol~~z:ttes wow t~v:rlwr:ltc~tl to dryness at reduced pressure and, aft,er taking up in water. the humin ww wntrifuged down and discarded. To remove thr dic:rrt)osylic wids from the tt!.clrolyznt,e, the solution was passed through a column of the txtsic resin, Amlwrlitc~ II<-113. A further separation was t,hrn made 1)~ passing the effluent Induced to :I small volume through a column of Dowes 50 of 2llGNo mesh in thr xcitl fomI :I( c9rtlitlg to 111~ method of Skin and AIoore (8). Samplw \vr’rr wllrcted in polo c~thylrnc~ planrhets using a fra~tioIl.collcctinF: :tpp:w:rtus of the I ylw tlrwrilwd t)y I,ic:n, I’r;trrson, and Greentwrg (9). For elution tlw follo~ving wcw usr~l ill t hc order given: wtcr, I .5 :md 2.5 S hydrochloric acids Iwth 0.3.5 satur:tte(l \vith sulfur dioxide nt room temper:lturP, :tnd 1 ,V antI 6 .V t1\~dro~hlori~* :tcills. The* sulfur dioxide was employed in :i lxtrtially successful at teml)t t 0 minimizct t hti fnrnxttion of met hionine sulfoxide. The acid was rcmnvtd from t hf s:ml~~lcs OII t hex pl:tnchets, :IIIII the radioactivities were drkrmined tl). counting using :t gastlow t utw. The n:tt,urr of the radioactive amino acids in thr various pwks \V:S determined Iry their position wid t)y running tno-tlimt~nsiori:tl chrnm:tt oprams oti :\liquots t:tk(ln from the peaks and comlxtring the travel of thf, rlinfl~driI~-l)osit ivc’ material and the radioactivity with st:rndards of :~pproprixtc amino xcitls. In addition. si~~~lr-tlime~lsiorl:L1 chromatogrnms of :tlicluots from alternate l)l:mch~~ts were m;& in orller to locate the nnnr:ttlio:~ctive xnino witIs. For c*hriml:ttog r:tl)hy. What man So. -I paper wws employed and either l)hr,rrol~-\\:ltcr, trr Ibut :11101 :twtir acicl mixtures. 13~ this means it w:ts found that thr amino witIs \VPI‘C c~lutwl from the> I)owex in the order given I)y Stein and iLlr)orr (N), witI1 tlw t~xwl)tiori that phrr~yl:d:mine appeared before rathrr than after arginine. fsolnlio/r oj’ (‘holine and C’reufine. Crr:ttinc \vas isolntc~tl in thr form of cr(y:It i tlinc pic.r.:ltcl from the aqueous cstracts of the tissues anal thr liwr i 101. Th~l l~ro(l~ urt was rwrystallized twice from the potassium picr:itc~ l)icric :wi(l rriistuw :trl~l its specific rndioactivit> drtermincd. Two mow rrc.r?st:llliz:ltiolls from thr same’ solvent \ver(’ then carried out :IS well :ts OIIP from w:ttcsr. .\ftctr r,:tc*h rwy!.st:1lliz:~ tinn thr spwitic :I.ctivity \v:ts rctletrrminrd. (‘holillc~ was isolated as thr chloropl;tt in:Lie from the :Llcotlnl :1ntl :LIw1IoI ether vstr:tc*ts of all the tissues coml)inc~d (11). After tlctt~rminstion of its slwcific* r:ldio:tctivity, :t part was degrxlrd to trimrthylxnille with :tlk:llinr~ ~wI~:III K:tn:ctp (12). The trimcthyl:tminr wxs also isol:lt<~(l as t ht> c.hlol,ol)l:rtitl:1t(. four t 111% det(~rmitiiLt ion of its CL4 content. Isolntir~rr 0.f Jlethionine nntl C’!ys!eine. In Fkpt :3 the sulfur-~l)llt:lirlirlg amino :wills wcr(. frwtiomttrd from aliquots of :t hydroiodic xcitl digest of 500 trig. of liver lnwtcin t)y the met,hod of Beach nnd Teque (13) in orclcar to avoid esccssivc~ losses t)y humin formation. From one aliquot thrl cuprous salt of cystcinr w:w prelwwd xnci from t,hr other the mixed salts of yysteinc x11(1 l~onioc~stc~it~r :w drscritwd t)>- thrsc xuthors. Ikr~rc~dation o,[ Alefhionine and Serinr. ilIet.hionint~ from thr al)l)rol)ri:lt(~ i’r:t(. tions from the column was degraded 1)~. digest,ion with h>-driodic xcid (11, 1.51 The mcltllyl iodide evolved was swept over into an :tlcoholic solnt ic)ll of t rimc~th~ I :Imine and isolated as t~etr:tmet~hylsmmonium iodide (161. Scrinc from the column was degraded with periwlic will. :III~ thcx formal111~ l~~.tl~ fornwl from 1he @-carbon was converted to the ~limwlon clcsriwt iw ( Ii I.
E. S. CANELLAKIS
450
AND HAROLD TARVER
RESULTS
Table I shows the amounts of Cl4 appearing in excretory channels following a single dose of methyl mercaptan to a rat. From the results it is evident that the methyl mercaptan in part escapesunchanged but that a large fraction of the dose is very rapidly oxidized to carbon dioxide. In the 6-hr. period of the experiment, about 40% of the dose appeared in the respiratory carbon dioxide. The results of Expt. 1 are substantiated by Expt. 3 in which mercaptan-P5 was employed. Again TABLE Distribution
I
of Excreted Radioactivity Following the Administration of Methyl Mercaptan-Cl* or P6
Expt. 1. Single dose of methyl mercaptan-Cl4 rat. Animal sacrificed after 6 hr. Expt. 3. Multiple (1.4 mg. total). Rat sacrificed after 8 hr.
Period
Expt.
1
(0.3 mg.) intraperitoneally to dose of methyl mercaptan-S36
Respiratory
carbon dioxide
hr.
%
o-l
29.2 6.2 3.8 1.6
l-2 2-4 4-6
Volatile
3
sulfur excreted
Fraction of dose excreted
O-l
6.4
l-2
0.0
Urine
O-6
2.3
Urine Total sulfur Total sulfate Inorganic sulfate
O-8 32.
31. 29.
a great oxidation is demonstrated. It might be anticipated that there should be some correspondence between the mercaptan oxidized to carbon dioxide and that appearing as sulfate in the urine. The observed discrepancy must be attributed to the fact that in one casethe mercaptan was given in a single dose whereas in the other the dose was divided, so that the last part was given only 2 hr. before sacrificing the animal. When the tissues were examined it was found that they retained considerable activity as shown by the specific activity figures given in Table II. It may be noted that in Expt. 2 the retention of the Cl4 by the proteins of the intestinal mucosa is’relatively lower than it is when
radioactive amino acids are administered to an animal (18). When the liver protein preparation obtained in Expt. 3 was washed with monot’hioet,hylene glycol (2-mercaptoethanol) about half of the activity was removed. Presumably this loss is due to the binding of methyl merc:aptalI as such to t’he protein rather t,han due t’o the introduction of a&vity from the protein into amino acid which in turn is iworporat’ed into the protein. Since the last dose of methyl mercaptan was gi\-en only 2 hr. before t’he animal was sacrificed, this observation is understandable 011 the basis of the results described in the previous communication (I). III Espt. 1 tbe label in the proteins from the animal was stable to monothioethylene glyc.01. Six hours after the administration, all t,he methyl mewnptnn may have heen oxidized. These results are similar t.o those
4
Live1 Kidney Spleen Lung Testis
17.5 11.1 9,s 11.5 8.5
Liver Liver”
10.1 4.9
‘I Liver protein preparation ous communicxbtion (l)].
Plasma protein Intestinal mucow Muscle Erythrocytes
washed with monothioet,hylene
22.7 16.7 2.2 0
glycol [see previ-
found by Lee and co-workers following the administrat8ion of cystine (19). When t(he wtivity of the cysteine and of the homocysteine-cystine from the liver protein (500 mg. with approx. 2500 counts/min. of 8”” stable to monothioethylene glycol) was examined, all samples had zero activity. It would appear, t’herefore, that the SS5associated with the protein in tbis experiment, was in the form of sulfate, and that the process lvhich leads to the formation of methyl mercaptan from methionine in rat, liver mitochondria is not reversible to any significant extent. The column radiograms of the hydrolyzates of the liver proteins of the rat, given methyl mercapt*a.n-P and of the mouse given L-methiol~i~w(“4H:j separated on I>o\res 50 are shown in Figs. I and 2, respeca-
452
E.
R.
CANELLAKIS
.4ND
H.4ROLD
TARVER
tively. It is evident from the first figure that Cl* from the mercaptan has been converted to serine (peak B) and methionine (peak h’), and from the second figure that the Cl4 of the methyl group of methionine has been converted to serine. In both experiments, a part of the methionine appeared as the sulfoxide (peak C), which occurred in approximately the same location as alanine. In both experiments, a remarkable amount of activity was present in the serine peak. In both radiograms 12,000 -
t I3
9,000 -
i ci cj 3,000 -
E A /\,L 100
k ti&---I.5
C
I 6 200 300 N. HCI t SO~----+2.5
Fraction
I 400 N. HCI HGI + SO,-
Number
FIG. 1. Column radiogram of the hydrolyzate of 1.36 g. of liver protein from the rat used in Expt. 2 after administration of methyl mercaptan-C”. Column: 113 X 1.4 cm. filled with Dowex 50, 2oOL4OO mesh in acid form. Solvents: water, 96 ml.; 1.5 N HCl with SO*, 470 ml.; 2.5 N HCI with SOZ, 420 ml.; 4.0 N HCl, 315 ml.; 6.0 N HCI, 220 ml. Location of peaks: serine, 204 ml. 1.5 N HCl with SO?; methionine, 260 ml. 2.5 N HCl with SO?.
there appeared small peaks, A and D, which could not be satisfactorily identified, although A may be due to aspartic acid. The similarity in these two radiograms is rather striking; they differ chiefly in that the administration of mercapt,an-C I4 leads to a protein richer in labeled serine and poorer in methionine than that obtained after methionineC14H3. Little activity was noted in the cystine peak, which may have been due to the loss of most of this amino acid in the form of humin.
METIIYL
MERC‘APT:iN
METABOLISM
-453
No significant radioactive peaks were found in those fractions not shown in the chromatograms. When the serine and methionine separated by the cbolumn radiography from the hydrolyzate of the liver protein of the rat given methyl mercqt,an-Cl-’ (Expt. 2) were suhjec+ed to degradation it was found, as shown it1 Table III, fhaLt bot,h the methyl group of methionine and t’hc 15,000 t
b+ I-$0+
B
loo 200 1.5N. HCI t So, --+
Fraction
300 2.5 N. HCI + SC+-
Number
IJrc;. 2. (‘olunm radiogram of the hydrolq-zate of 0.16 g, of liver protein from tlui mouse used in I
@c*arbon of serine were highly active. The results also show t,he high wti\‘ity of the choline isolated from the carcass of the same animal. Moreover, the activity of the trimethylamine accounts for most of that present’ in the choline. There may be some activit,y in the ethauolamine portion of the molecule due to decarboxylation of serine, which has becwme labeled in the p-carbon. Creatinine from t,he body creatine was also c1uit.e a&ve.
354
E.
S.
CANELLAKIS
AND
HAROLD
TARVER
It is obvious from these experiments that the metabolism of the methyl group of methyl mercapt’an is in some respects similar to that of the methyl group of methyl alcohol as shown by a comparison of the results reported here with those obtained by V. du Vigneaud and coworkers (20, 21), and that the results are explicable on the same basis, namely oxidation of the methyl group t’o an intermediate which may either appear as the P-carbon of serine or may be reduced again to reappear as a labile methyl group. TABLE Distribution
III
of Activity
Ezpt. 2. Multiple after 10 hr.
in Vutious Constituents of the Rat Administration oj Methyl Mercaptan-Cl4 dose of methyl
mercaptan-Cl4
(1.9 mg.
total).
Following Rat
the sacrificed Specific activitya c0unfslmin.l +w.
Methyl
group of methionine from liver protein isolated as tetramethylammoniumiodide................................................. &Carbon of serine from liver protein isolated as dimedon. Creatine isolated from tissues as creatinine picrate. Recrystalliaationsh l.......................:............................ 2 .._..._...__.................. .._... .._........ 3.. Choline isolated from tissues as chloroplatinate. Trimethylamine chloroplatinate from degradation of choline above.
24.7 108.5 20.3 20.3 20.5 179. 187.
a Values given are not comparable because they are not on a molar basis and are not corrected for self-absorption. Corrected activities would give no additional information because of the large dilutions with the preformed compounds largely unknown as to amount and turnover rate. b See under Methods for details. SUMMARY
In experiments in which methyl mercaptan-CL4 or S35had been administered to rats in vitro the following has been found: 1. Both the carbon and the sulfur of the mercaptan are rapidly oxidized to carbon dioxide and sulfate, respectively. 2. The methyl carbon of the mercaptan is converted to the p-carbon of serine and methyl groups of methionine, choline, and creatine. 3. Sulfur from methyl mercaptan does not appear to any significant extent in the methionine or cystine of liver protein.
METHYL
MERCAPTAN
435
METABOLISM
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