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940 CHARLESD. ROBESON ND JAMES G. BAXTER Vol. 65obtained from active fish tissues by acetone treat-ment.

The solubility of the factor in dilute salt so h-tions, the rapid destruction by heat, and the pre-cipitability by common protein precipitants indi-cate the fish principle to be of protein nature.The destruction of thiamine is maximal at PH

9.1 and 60", is proportional to the amount of prin-ciple used and is characterized by first-order ve-locity constants. These facts together with theevidence of the catalytic nature of the reactionstrongly suggest that the principle effecting thethiamine loss is an enzyme.ROCHESTER,. Y. RECEIVED PRIL 6, 1943

[CON~RIBUTLONNO. 7 FROM THE LABORATORIES O F DISTILLATIONRODUCTS, rNC.1a-Tocopherol, a Natural Antioxidant in a Fish Liver Oil

BY CHARLES . ROBESONND JAMESG. BAXTER'This paper describes the isolation of a n atu rd

antioxidant from shark liver oil (Mangona, fromBrazil; 3800 units of vitamin A per g.) and iden-tifies it as natural a-tocopherol (hereafter theterm, "tocopherol" means "natural tocopherol").Evidence is further presented to show that a-toco-pherol is the major antioxidant in the oil. Thisfinding is of interest because it indicates that thetocopherols may serve as natural antioxidants infish liver as well as vegetable oils. Conflictingevidence has previously been reported on the oc-currence of vitamin E in cod liver oil.*t3

Mangona shark liver oil was used in the investi-gation because the livers were available and couldbe processed in this Laboratory. A tocopherolconcentrate was also prepared from soupfin sharkliver oil (Eugaleus Galeus, 94,400 units of vitaminA per g.), but since the oil was obtained commer-cially, it could not be proved that the tocopherolhad not been added during processing, e. g., byusing some vegetable oil to extract the livers.Isolation of Antioxidant.-The isolationmethod employed was based on the hypothesisthat the antioxidants in the liver oil would, likethe tocopherols in vegetable oils, be concentratedby molecular distillation in the more volatilefraction. Therefore, the oil extracted from thelivers was distilled and the fraction distilling be-low 225' at 0.003 mm. pressure was collected.The antioxidant in this distillate was detected bymeasuring the activity of the fraction in protect-ing vitamin A against atmospheric oxidation.The test method was as follows.A solution of vitamin A in a substantially anti-

(1) Presented before the Division of Biologicnl Chemistry of the(2) Sure, . Bid . Chcm., 74, 45 (1927).(3) Nelson. Ohrbrck. J n a ~ r od Taylor. Am . J . Phrr iol . . 16. 47 6

Amer i can Chemical Sodety, Buffalo meeting, September, 1942.( m a ) .

oxidant-free oil (5500 units per g.) was preparedby dissolving crystalline vitamin A in distilledsardine To aliquots of this (3 cc.) was added5% of the undistilled shark liver oil, 5% of thedistillate, and 5% of the distillation residue.These samples together with a blank sample con-taining no added fish liver oil were exposed toair, in thin layers, a t 55' for two and five hours.A t the end of each heating period, the vitamin Apotency was determined by the antimony tri-chloride method. The vitamin A recoveries aregiven in Table I.

TABLEANTIOX~DANTCTIVITYOF MANGONAHARK IVEROIL

FRACTIONS

SampleSolution of crystalline vitamin A inNo. 1 + 5% Mangona shark liverNo. 1+ 5% M.S.L.O. distillateNo. + 5 % M.S.L.O. residue

distilled sardine oil (6500units/g.)oil (M.S.L.O.)

Sp o! originalvttauun A po-tency after ex-posure to air at55 " for2 Hrs. 5 Hrs.31 1058 2595 9141 20

I t was concluded from the data that the sharkliver oil contained an antioxidant (or antioxidants)which was concentrated in the distillate since theresidue had little antioxidant activity. Subse-quent isolation work was, therefore, confined tothe distillate which contained glycerides, vitaminA esters, and sterols as impurities. The methodfor separating the antioxidant from the impuritieswas based on the hypothesis that it was a phenoliccompound like the tocopherols.

The distillate (11, Experimental Part) was es-terified with succinic anhydride. The half suc-

(4) Distilled sardine oil was prepared by atripping sardine oil in amolecular still and discarding the 6rst 15% containing natural anti-asidant., T ~ Rex t 50% of the distillate w~pnhe dillrent wed.


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May, 1943 a-TOCOPHEROL,NATURALNTIOXIDANT IN A FISHLIVERO ILcinates were separated from vitamin A esters andglycerides by neutralization in absolute alcohol,dilution with water to 83% ethyl alcohol, and ex-traction of the non-hydroxylic constituents withpetroleum ether. After acidifying the alcoholsaponified, and the sterols were separated by crys-

1.81.6

layer, the half succinates (IV) were recovered, 1.4tallization from ethyl formate solution a t ow tem- .@ .2peratures.he filtrate Andeddishistributedil (VI) betweenas recoveredetroleumrom 41l.oether and Byo queous ethyl alcohol. 8 0.8alcohol layer. The petroleum ether fraction (VII) 0.6was then fractionated by adsorption on Floridinearth.6 After elution an antioxidant concentrate 0.4

0.2VIII) was obtained as a reddish-yellow oil,tivity as 5% of the distillate (Table 11).

Free vita-inin A plus some of the antioxidant passed into the

0.048Yc of which had the same antioxidant ac-

941

II1I

'

*

8 ,-\0 : ;

, \

.,

I

1,. ,' , I

, , ,'* :. ,

't,' . _ I

1.81.61.4

ACTIVITYF ANTIOXIDANTONCENTRATERO M MANGONASHARKLIVER IL

7" of orimnal vitamin A w-

'

'

.

Sampletincy after exposure to aiiat5 5 O for hours2 4 7 9 1 0 1 1

1. Solutionof crystalline vita-minA in distilled sardineoil residue (5500 units/g.) 31 10(a) No. + 13%Mangonashark liver oil distil-late (11) 89 93 82 8f L 60

2.

(b)No. 1 + 5% I1 95 91 75 72 63(c) No. 1 + 1.75% I1 94 87 69 62(a) No. 1 + 0.125% anti-oxidant concentrate(VIII) 96 93 85 84 693 . (b) No. 1 + 0.048%VI11 95 89 77 74 64(c) No. 1 + 0.017% VI11 89 81 63 55Concentrate (VIII) had these properties char-

acteristic of the tocopherols: (1) It gave a redcolor when heated with nitric acid in alcohol solu-tion. (2) By the tocopherol assay method ofEmmerie and Engel,6 it gave a red color corre-sponding to 78.5y0 tocopherol. (3) It had anultraviolet absorption band a t 292 mp similar tothat of a-tocopherol (Fig. 1).A crystalline palmitate of the antioxidant wasprepared from VIII. The crystals (laths, m. p.40.5-41.5') appeared to be identical with those ofnatural a-tocopherol palmitate (laths, m. p. 42-43O).' The carbon and hydrogen analysis sup-

(5 ) Floridin earth (Floridin Company, Warren, Pa.) is a specialgrade of fuller's earth recommended by Emmcrie and Enpel I&c.t w o . ch i n . , 68, 28 3 (1039)l for the removal of carotenoids.

(6) Ernrncrieand Engel, Rec. fro%chim., 6?, 1351(1938).(7 ) Baxter. Robeson, Taylor and Lehman, Tms JouwrAL, 66,918 (IW).

Fig. 1.-Comparison of absorptionspectra of :- nti-oxidant concentrate (VIII), E &z, 92 mp = 62.5), and- - - - - natural a-tocopherol, (E 292 mp = 73).ported this conclusion. The absorption spectraof the two esters and their extinction coefficientswere nearIy the same (Fig. 2). The vitamin Eactivity of the palmitate was determined by Dr.P. L. Harris and M. H. Joffe of this Laboratory.

.g1.21.0

8 0.8.eY0.60.40.2

I I2 0 0 2 4 6 8 3 0 0 2 4Wave length (mp).

Fig. 2.-Comparison of absorption spectra of :- al-mitic ester of antioxidant, ( E:z. 286 m p = 28.1), and- - - - - - natural a-tocopherol palmitate, ( E :& 286 m p =26.8).


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942 CHARLES) ROBESONAN D JAMESG. BAXTEK Vol. 55'The ester was found to be equivalently as activeas natural a-tocopherol.* I t wits concluded thatthe an tioxidant isolated from hlangona sharkliver oil was a-tocopherol.

Evidence was obtained indicating that a-toco-pherol is the major antioxidant iii this oil. Toprove this, the antioxidant activity of three con-centratioris of the liver oil distillate (11, 1.73-137)) as compared with three concentrations ofthe a-tocopherol concentrate prepared from it(1'111, 0.017-0.125(?&) (Table 11).

I t appears from the data t hat EiyOf I1 had sub-stantially the same antioxidant activity as 0.048%of "111. Thus, if 5% of I1 contained the sameamount of a-tocopherol as O.O$S% of VIII, itwould seem justifiable to conclude that a-toco-pherol is the principal antioxidant in the oil.The percentage of a-tocopherol in I1 could not bedetermined directly because the large amount ofvitamin A present interfered. However, VI, ob-tained from it and containing orily a small amountof vitamin A , could be assayed by the Emmerie-Engel procedure and the percentage of a-toco-pherol in I1 calculata. From 159 g. of I1 wasobtained 3.35 g. of VI containing 39.4y0 -toco-pherol, corresponding to 0 . 8 % a-tocopherol in11. Therefore, 5cTc of distillate I1 containcd thesame amount of a-tocopherol as 0.053'7, of VI11(78.5y0 a-tocopherol), which agrees well with thevalue 0.04S%.

We, therefore, conclude th at a-tocopherol is themajor natural antioxidant in Mangona sharkliver oil. It was calculated that the fish liver oilcontained about 0.01% a-tocopherol, which isabout one-tenth as much as is present in unre-fined cottonseed oil.

From soupfin shark liver oil by th e same proce-dure a concentrate was obtained containing 76.7%tocopherol by Emmerie-Engel assay. The ultra-violet spectrum of this preparation had292 mp = 77, a value greater than t hat of pure a-tocopherol. This indicated the presence of ex-traneous absorption. The concentrate was notfurther investigated for reasons previously stated.The percentage of tocopherol in the original fishliver oil was calculated to be approximately o.0470.

ExperimentalIsolation of Antioxidant from Mangona Shark Liver Oil

Extraction of Oil from Livers.-The livers (34.9 kg.)were ground in a meat grinder. The slurry wa s mixed(8) Harris and Joffo, Tara JOO~NAL,U, 82 6 (1943)

with 1.5 volumes of water coutaining sodium hydroxide(698 e . ) . The mixture was blown with steam for fifteenminutes arid the oil layer was allowed to separa te. Afterwashing with hot water and centrifuging, the liver oil(1, 15.55 kg.) was obtained.

Distillation.-The fish liver oil (I)was distilled in amolecular still and the more volatile fraction distillingfrom 145-240" at 0.003 mm. pressure was collected (11,159 g.). In collecting this fraction, the temperature wasraised in 10' intervals and two cycle< werc taken at eachtemperature.

Esterification.-The distillate (11) n pyridine (400 c.)containing succinic anhydride (5 7 8 . ) was heated for twohours at 70'. The solution was then poured into dilutehydrochloric acid, extracted with ether, and the extractsuccessively washed with acid and water. After drying,the solvent was distilled, leaving a red viscous oil (111,1739.).

Extraction.-The esterified distill ate (111) n absoluteethyl alcohol (1200 cc.) was neutralized with 2 N aqueoussodium hydroxide. Th e solution was adjusted to 837;,alcohol with water and ext racted with four 450-cc. portioiisof petroleum ether. The alcohol layer was acidified with57, hydrochloric acid and extracted with ether. The ex -tract was washed with water, dried, and the solvent wabdistilled. The impure antioxidant half succinate was aviscous red oil (IV, 73 9,) which solidified at room tempera-ture.

Saponification.-The acid succinate (IV) as saponifiedby a procedure previously described.' The ester in abso-lute ethyl alcohol (700 cc.) was refluxed in an a tmosphereof nitrogen for ten minutes to iemove dissolved air, andaqueous potassium hydroxide (70 cc. concentration, 50 g.per 100cc.) was added. After refluxing for thir ty minutes,the saponification mixture was diluted with water andextracted with ether in the usual way. After distillationof ether under reduced pressure (r .p.), a tan-colored solid(V , 15.59,) was obtained.

Removal of Sterols.--The concentrate (V) n ethylformate (I50 cc.) was cooled to 5" for fifteen hours andfiltered to remove sterols (11.7 g.). The solvent in thefiltrate was distilled (r . p.), eaving a dark red oil (VI, .359,) ontaining 39.4y0 ocopherol by Emmerie-Engel assay,

Distribution.-Fraction VI, in petroleum ether (100cc., b. p. 30-65'), was estracted four times with 100-cc.portions of 83% aqueous ethyl alcohol which had beensatu rate d with petroleum ether. Solvent was removedfrom the petroleum ether fraction by distillation ( r . p.),giving a viscous oil (VII, .2 9.) containing 5970 tocopherol.

Adsorption.-Fraction V I1 was adsorbed froin petro-leum ethe r (15 cc.) on a column (9" X 12 mm.) of Floridinearth. After development with petroleum ethe r (80 cc.)two ban ds were observed. The upper half of the columnwas bluish-green, the lower half was colorless and containedtocopherol which was eluted with ether containing 5%methyl alcohol. The eluate was freed of solvents by dis-tillation (r . p.). The residue, a reddish-yellow oil (VIII,0.59 g.), contained 78.5y0a-tocopherol by Emmerie-Engelassay and had 292 mp = 6 2 . 5 .

Antioxidant Palmitate.-A portion of VI11 (0.28 e.) inethylene chloride (2 c. ) and pyridine (1 cc. ) was treatedwith palmitoyl chloride (0.9 cc.), in ethylene chloride


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May, 1943 THEACTION F SODIUM(2 cc.), at room temperature. After standing a t roomtemperature for twenty hours, the reaction mixture waspoured into ether and the ether extract was washed suc-cessively with di lute hydrochloric acid, 0.5N aqueous potas-sium hydroxide and water. The ether was removed bydistillation leaving a white solid containing free palmiticacid. Palr:.itic acid was removed by adsorbing the crudeester frotn petroleum ether (15 cc.) on a column of Doucils(9'' X 12n .m. ) . 'The column was washed with petroleumether (7B cc. ) and filtrate and washings were evaporatedleaving a residue (0.302 g . ) which was crystallized frornethyl alcohol (6 cc.) at 5 " . The antioxidant palmitateformed lath-like, white crystal s (0.18 g.), m. p. 40.5-41.B0,which were identified as a-tocopherol palmitate.-

(9) Doucil (American Doucil Company, 121 South 3d Street.Philadelphia, Pa.) is a sodium aluminum silicate.

ETHYLN ( --)~-BROMO~CTANE 943Anal. Calcd. for C4hH~03:C, 80.76; Ii , 12.06.

Found: C, 80.81; H, 11.91.summary

A natural antioxidant has been isolated fromMangona shark liver oil and identified as naturala-tocopherol. Evidence was obtained indicatingthat a-tocopherol is the major antioxidant presentin this fish liver oil.

This finding is of interest since it indicates th atthe tocopherols may act as natural antioxidantsin fish liver oils as well as in vegetable oils.ROCHESTER, EWYORK RECEIVEDEBRUARY, 1943

[CONTRIBUTIONROM TH E FRICK HEMICALABORATORY,RINCBTQN NIVERSITY]Reactions of Organometallic Compounds with Alkyl Halides. I. The Action ofSodium Ethyl on (-)2-Bromooctane

BYNORMAN . BRINK,^ JOHN F. LANEAN D EVERETT. WALLISPrevious studies2 in this Laboratory have

shown that the Wurtz reaction of an optically ac-tive alkyl bromide with sodium leads to a com-pletely optically inactive product. Thus, when(+)2-bromobutane is treated with sodium a troom temperature, the resulting 3,4-dimethyl-hexane shows no detectable optical activity.This result would be consistent with a formulation3of the Wurtz synthesis involving initial formationof free radicals

Na + R X+ ' + NaXfollowed by their combination to give the ex-pected coupling productor by their disproportionation to alkane and al-kene by-products

2R' ----f R H + olefin.Secondary alkyl radicals would be expected toshow extreme optical instability, since they pos-sess numerous planar resonance states comparablein energy to the normal state. In this they re-semble the corresponding carbonium ions moreclosely than the corresponding carbanions, theplanar resonance states of which are considerablyhigher in energy than the normal state.4

(1)

2R'+ -R (2)

(3 )

(1) Sayre Fellow in Applied Chemistry, 1942-1943.(2 ) Wallis and Adams, Tms JOUKNAL,6, 38.98 (1933).(3 ) Cf.Hbckel, Kraemer and Thiele, J . prakl. Chcm., 14% 207(1935); Bachmann and Clark, TRIBJOURNAL,9, 2089 (1927);Richards, Trans. Faradcy Soc., 86,956 (1940).(4 ) Baughn. Evan. and Polanpi, Trans. Faraday Soc.. 81, 87 7(1941).

More recently,6sodium alkyls have been shownto act on alkyl halides in a manner typical of thesalts of very weak acids, either to effect substitu-tion of the carbanion for halogen

(Na+)RI- + R2X+ Na+)X- + R1- R1 (4)or by their strongly basic action to remove theelements of hydrogen halide with the formationof an olefin(Na+)R,- + R-CHZ-CH~X -In addition, when X is bromine or iodine, metal-halogen inter~hange~~

NaRl + RzX --+ NaRz + RIXcan occur. It has been further suggestedsb thatthe initial stage of the Wurtz synthesis may in-volve direct formation of a sodium alkyl

2N a + R X -+ NaR + N aXand t hat subsequent stages of the reaction involvethe action of the sodium alkyl on additional alkylhalide according to equations (4) and (5). Ifthis be granted, the observed products of theWurtz synthesis may be accounted for without re-course to the concept of free radicals as criticalreaction intermediates.In order to account for the complete optical in-activity observed in the formation of 3,4-dirnethyl-hexane from (+)%bromobutane and sodium,however, such an interpretation must be ampli-

RIH + R-CH=CHZ + (Na+)X- ( 5 )(6)

(7 )

(5) (a) Whitmore and Zook, Tmn JOW~NAL, 64, 1788 (1942):(b) Morton, Davidion and Haksn, ibid., 64, 25142 (1942).

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