Isolation of vitamin K from animal tissue

.\ltCHIVES

OF

BIOCHEMISTKT

Isolation

Department

ANI)

123, 514-530

BIOPHYSICS

of Vitamin

JAI1153

W. HAI\IILTOY

of Biochemistry,

University

K from I1?JD

of Louisville, Received

(1968)

July

Animal

R. DUSC,AS School

Tissue DALLAM

of Medicine,

Louisville,

Kentucky

31, 1967

The isolation of phylloquinone from beef liver has been attempt,ed. IIevelopment and use of a biological assay system has demonstrated that beef liver, beef liver lipid extracts, beef liver mitochondria, and the beef liver mitochondrial lipid extracts contain some compound that can alleviate elevated prothrombin times produced in chicks by a vitamin K-deficient diet. ,111 attempts to isolate and identify this activity have been unsuccessful. The adoption of a modification of an isolation method developed by Fieser [J. Am. Chem. Sot. 61, 3467 (1939)] facilitated the separation of two compounds from rabbit, chicken, and swine liver, one of which has been tentatively identified as a phylloquinone. The highest concentration was observed in rabbit liver (5 rg/gm lipid extract), and chicken liver contained a lower conrentration (3.33 hg/gm lipid ext,ract). Although absolute proof of the structure must await further work, the evidence thus far suggests that the compound may be Z-methyl-3A6-dehydrophytyl-1,4-naphthoquinone. The data obtained on the second compound isolated from rabbit, chicken, and swine liver do not permit any conclusion to be reached at present, but its spectral properties appear to be identical with those of or-tocopherolquinone.

One of the problems we have pursued is the exlucidatjion of the mechanism of action of phylloquinone in biochemical systems. The suggestion that quinones function in t’he respiratory chain has been made by a number of workers ( 1-I). Phylloquinone, vitamin E, and more recently, ubiquinone, have all been considered as serving some role in oxidat,ive phosphorylation. These three structurally similar quinones are easily oxidized and reduced, a property favoring participation in biological oxidation-reduction reactions and in particular mitochondrial electron transport. At the present hhere is strong experimental evidence that ubiquinone has such a role, but evidence on the other two compounds is poor. 1 This work was supported by grants from National Institutes of Health HE-02102 and National Science Foundation GF-17427. *The work presented was submitted to the (+raduat,e Faculty in November, 1965, in partial frdfillment for the degree of r)oct,or of Philosophy. Present address: Veterans hdmirlistraiiotr 111~s. pital, Kansas City, Rlisso\ui.

That phylloquinone does have a definite biological role is well established. It is knowII to be necessary for the biosynthesis of t,he proenzyme, prothrombin (5, 6). The exact nature of the phylloquinone requirement, is not laow11, but it is generally felt that t,he vitamin may be a necessary component of the enzyme system leading to prothrombin synthesis (7). The possibility that phylloquinone is involved in electron transport was first suggested by :\ lartius (1) when he observed that mitochondria isolated from vitamin T\deficient, chick liver had a reduced capacity for the synthesis of adenosine triphosphate. l\Iore recently, Rrodie (4, S-IO), working lvith a bacterial system, has obtained evidence that. phylloquinone may be required for elect’ron t,ransport and oxldative phosphorylat,ion in Xycobacteriun~ phlei. l’hylloquinonc has been isolated in pure form from alfalfa (II), and bacteria (12-1.5). With respect to the animal kingdom, no phylloquinone has yet been isolat,ed, although indirect, evidence for the occurrcncc of such :I compound in animal tissues has

ISOLATION

OF

VITAMIN

been reported. Martius and Esser (16) found a compound in lipid exkscts of chicken liver with a partition coefficient similar to that of one of the menaquinones. There have been no conclusive data, however, on the fype of Ii vitamin that may occur in :mimal tissue. The present investigation is concerned mith the occurrence of phylloquinone in animnl mitjochondria and liver tissue.

Fresh hoviJrc liver was obtained from Ihe Fisher Parking Company, Louisville, KcJltttcky, iJnn1ediately after removal from the animal. The liver was placed in rrrtshed ice, lrarrsported to the laborat ory , and stored in a rold rcwJn (2-J”) where all subsequent operations were performed, tt11less other&c noted. I~rsrtally o~lr liver (15 po~tt~ds~ was processed at :1 t imr. The tissue was first trimmed of fat, sliced iJJt0 small pieces, and cc,Jlvertcd to paste ill a~1 electric griJrder. The paste was hontogeJ1ixed in 0.25 11 sucrose with a LVariug Blendor (model MR-3) lo yicl(l a 10% homogenate (TV/V). lSleJ1ding \v:Js tlo~rr at, a speed of 15,000 rpm for 2 Jnintltes, after which the homogerlate was filtered throttgl1 two layers of l”\‘o. 40 c~hcrsrcloth to remove debris. The filtrate was sttbsequently frozeJ1 in one-half iJ1ch layers iJ1 Kmiurh diatneler baking tins. Tl1is was tlorlc to fnrililnlr lyophilizstiott it1 the model lo-104 I7irtis freeze-dryer. After the Jnnterial was dry it was groltnd to a fiJ1e powder with a mortar and pestle. In later work, lyophilizcd chirkcJ1, swine, :11td rabbit livers were prepared wi(hout hoJnogrn:~t,iotr. The fresh t issrte was ground iJ1 a11 c~lectric griJider a11d frozen directly for lyophilizatiot1. The dried material was also grottnd to a powder. lSoL.,‘rIoN

OF l3EEF

LIVEIt

P\II’tY~CHONt)RI.\

The procedure employed for isolation of liver rnitochoudria xas sinlilar to that dcwribed above for the preparation of Iyophilixed whole liver homogenat,e. Elevcu porrnds of ground liver was homogenized in small batches wilh 0.26 .II sltcrose in n WariJJg Blendor at 15,000 rpn1 for 1 minltte. After thr hotnogenatc was paswd throttgh two layers elf No. 40 rheesecloth, the filtrate 1120 litrrs I’roJn 12 kg of liver) ml ovcrJ1ight irl the c(~ld rooJ11 to precipilate thr heavier particles. The lromogeriate was centrifltgetl in a Lotudcs “Bet:thge,” Jnodel A) by 11sing ihe coJltiJlllollS-fl~~W syslern wit11 i hc CFIG1 rot or. The flow raic was atlj~tstctl 1r1 fwd iJrt,o the rollwt iotr bottle at the

K FROM

LIVER

51.5

rate of 30 ml/miirutc. The first cetitrifrtgstioir removed uuclei aJ1d cellular debris and was made at 1820g. CentriftJgation of the resultitlg srtpernat,ant fraction was performed at 15,200g arid yielded a thick tan mitochoridrial residue. The isolated mitochondria were resuspetlded in 0.25 M sttcrose with the Waring Blendor at, 3600 rpm. The suspension was receJrtrifuged xl 15,200g to yield washed t1titoct1oJrdria. They were suspeJldetl in a mi1rimutn volume of 0.25 %t sucrose with the Waring Blcndor. Freczc-drying was rarried out in :I tnanner similar to that. described for \vhole GssJte homogeJrnt,e. After drying was eornplcte the Jnitochondri:J were ground to a fine pcnvdcr.

CHICK had

The basal diet” the followirlg

DIET

used iJJ all of the chick perccJ1t:cgr~ compositioll:

Commercial soybeair oil meal, (~:rouJtd yellow corn, CaCO2, CaClu, CaIII’O4, Fortified soybca11 oil,4 B-vit,nniiJi premiq5 RIlrSOa, ZnCOs,

ass:~ys

:32 62.2 1 .3 0.5 2 I I 0.025 0.01

To insure a miniJnurJ1 of intestitlal flora in the ctlicks, 150 JJlg st1lf:tclttiJlo~;lillr (SIcrck, Sharpe aJ1d Dohme) was added to 100 gtn of diet. AftcJ preparation, the dry ingredients :rJld fortified oil were stored in the cold room. Jltst prior LO I he beginning of au experiment the two were Jnixed in an amount sufficient for 1 week’s supply of diet, which was also stored in the rold room. (After 3 weeks of storage, barteriologiral esatni11atiorl rcvexlcd that growth of Jnirroorg:tJ1isrn in thr diet, 3 The diet was kirldly provided hy 1 jr. Boyd L. O’l)ell, 1JJiiwrsity of hlissouri. 4 The fortified soybeaJ1 oil was prepared t,y mixing the oil (Ralstorr-I’ttrina Company, I,ouisville, KY.) with the appropriate :rmoJt11t of WhiIr’s Cod Liver Oil CotrceJltrate so that 1 gm of the final mixture rontairird 1200 16 of vitamiu :\ nJ1d 100 IG of vitan1iJl D. Alpha Iocr,pheryl :wet:ttc~ was added so that rnrh gJ’ar11 of the Jnist~rJre roI1tail14 2 Jllg. 5 ‘The 1%vitamiJ1 prc~J1Jix xv:w pr(~parctl 1)~ Jrlisitlg t hc arighed B Vit:iJllillS wit11 :l ml:111 nmouJlt of thtl aoybcan oil meal. One> g11l of the fi1rnl nlis(rare sl1pplied t,he folloaiJlg: 0.14 mg riboflavin, 0.88 JJlg ralciuni paJ~tothet1:J1e. 212 rng niacitl, 21 Jng rholin dihydrogeu citratr, 0.60 pg vit:tttJiJl lj312r 0.44 JJlg proc:iiJle lJeJJicillit1, a11tl 0.1 gJ11 JJlethiotJinc.

516

HA%ZILTON

AND

was negligible.) The water given t,o the animals also contained sulfaquinoxaline, 8 gm/20 liters. The cages in which the animals were kept were washed two or three times a day, depending on the nllmber of chicks orcupyitlg each tier, to prevent access to feces.

The same general procedllre was used for extraction of lipids whether the material was lyophilized whole liver or lyophilized liver mitochondria. (All solvents used for extract,ion were redistilled and examined spectroscopically before used.) In a Soxhlet extraction apparatus, the powdered material was refluxed with chloroform-methanol (2:l) at 54” for ti hours (250 ml solvent/20 gm of tissue). After removal of the solvent with a heavy,duty rotary evaporator, the resulting brown oil was redissolved in petroleum ether (4@50” b.p.) and washed twice with water. It was determined that about, 20’% of t,he dry tissrre and 30% of mitochondria was lipid. In instances where the total lipid extract was not, required, the petroleum ether solution was added to 3-4 volumes of cold acetone and placed in the cold room for several hours. The precipitated phospholipids were removed by suction filtration. Roth residue and filtrate were stored in amber bottles in the dark after removal of the solvent. With whole chicken, swine, and rabbit liver the extractions were performed by rapidly stirring the powdered material with the solvent at a ternperature of 50” for 2 hours (500 gin of tissrle/3 liters of chloroform-rnetharlol, 2:l). Three changes of solvent were required to remove all the lipid. After evaporat,ion of the solvent, the brown oil was dissolved in a minimum amolnlt, of petroleum ether, and 3-4 volumes of cold acetone were added to remove phospholipids.

Prothrombin time determinations were conducted initially by tlsing a modification of the method of Quick (17). A simplified micromethod was developed and used for the majority of the determinations to be reported. The procedure utilized a special instrument” which had the combined features of providing constant temperature, constant shaking, and the use of multiple micro-samples, usually consisting of abollt 0.06 ml whole blood.’ The l-cc tubercldin syringes and 6 Designed and manufactured by Mr. Sam Adams, University of Louisville School of Medicine, Louisville, Kentucky. i Details of the method and inst,rument will be provided on request to Dr. I<. I>. Dallam.

DALLAM the 25.gauge, l-inch needles used to collect blood from the animals were siliconized regularly with a water-soluble silicone preparation. The blood (0.25 ml) was withdrawn from the chick by direct heart puncture and used immediately. With normal prothrombin times, duplicates usually agreed within 5 seconds, but with deficient blood the difference was as much as 12 seconds. CHICK

ASSAY

PROCEDWE

One-day-old, White Rock chicks were obtained from Crayden’s Hatchery, Itamsey, Indiana. The animals were divided into several groups of 1-8 chicks, and the number of grotlps was determined by the number of substances to be tested. Each animal was marked with a leg band. Two control groups were always maintained for each experiment. One of these received the vitamin K-free diet plus a daily oral supplement of phylloyuinone, and the other control group received only the deficient diet. Thus it could be ascertained whether the diet was functioning properly and whether the deficiency could be correcbed by phylloquinone. The remaining groups were fed the deficient diet. Prothrombin times were measured at 3-day intervals to follow the development of the deficiency. After 12-15 days, when the animals showed elevated prothrombin times, the experimental groups were given the substances to be tested. When these substances were solids, as in the case of the lyophilized whole liver homogenate or the lyophilized mitochondria, they were mixed with the diet in a 1:l ratio and fed to the chicks ad li6itutn. When the material to be tested was an oil, it was administered orally in the amount of approximately 150 mg per day per chick. [Seegers (18) has reported that 5-8 pg phylloquinone can cause the elevated prothrombin time of a deficient chick t,o return to normal.] Once the animals were started on t,he supplements, the prothrombin time of each animal was determined every 2 days.

Ultraviolet spectra were obtained by using a Beckman DK-2 ratio recording spectrophotometer. Standard quartz cells with a l-cm light path were used in all determinations. The instrument was operated at room temperature, and unless stated otherwise, the solvent was spectroqualit) cyclohexane.

I’oper chromulography. Reversed-phase partition chrotnatography was utilized predominantly. Whatman So. 1 chromatographic paper was impregnated with petrolatum by soakittg 3.9 X

ISOLATION

OF

VITAMIN

X-cm strips iI1 a 10% solllt,iou of pelroler[m ether (w/v) for 10 miurltes, followed by air-drgiug. The descending olre-dimensiorlal techniqrle was enlploypd wilh standard chambers 1,~ losing an mixture. I)evelopn-plopanol~n~tt.er (4: 1) solvrrit ment of the chromatograms took abollt 16 hours. Thin-lo!pr chrornaloyruphy. A 1)esnga No. Ii00 thill-layer chromatographic apparatus was IWXI with glass plates prepared u-ith silica gel (; ac~~,rding to Stahl (19). Ascending development ether-diethyl was c,arriPd out 111 a petroleum rt,her (95:5, v/v) solvelit system or belIzelie. 1 )evclopmeut was for GO miuntes. (.‘o/~trtn chromatography. Standard t.echuiqucs for cwlurnr~ chromatography of lipid materials were employed. The adsorbellts rlscd throughout thr work xere Decalso and silicic acid (lipid chromatography grade). Glass C~O~~ITIII~S of variorls pizcs provided with a disc of sint;ered glass to RlLpport the ndsn+ellt aud Teflo11 stoprocks were IlSfVl. MWIIOI~S

OF I)ETECTION USED CHROMATOGR \PHY

IN

Several methods of different sensitivity aud specificity are currently iu llse for the detection of quinoncs on rhromatograms. The underlying prprequisite for utilization of t,hese methods is ~0 have a sample which is relatively pure. Thrls thrsr procedllres were not employed when working \vith total lipid extracts, b\lt only after S\IP~ PStracats hnd i)cett sllbject.4 to clt.her ptlrificntiou nlethods &signed to remove utiwauted lipid matc,rials. QII~IIOIIPS iu gpuera! may I)e detected on paper strips or thin-layer plates by the qllenching of their fll~orrscrnce rulder IIltraviolct light. This n~rthod is rather illselrsitive since at least 15-20 rg is rect\Liretl for defitlite observation. This method wis Ilot considered Iiscful on imprlrc fractiolrs. The rnethotl of dciectiou that best f\llfilletl the reqllirements of sensitivity alltl a1)rcificit.y was cme developed by I,ester and r::Lt~l;is;trrna (20) for the iibiquinolle series rlt ineotetraeolillm chloride. The lizillg tllc (ly?, ],hv,yr limit. of tlPtectiol1 with this method is 1-5 pg of ph~lloc~llillclrle or mel~:lclllil~otlP~1.

Tllis procrd~ue was atlapt,rd from Fieser (21)) \~-ho tlrveloped it while working on t,he isolat iot1 of I)lrrlloquillolle from spinach leaves. It, was late1 llsetl I,, the isolation of phylloqrIirrone from r)t bet I)lalrt materials. :\ 5.gm portion of the total lipid extract from ~~-lli~tl t hc majority of phospholipitl had been re-

K FROM

LIVI:H

.ilT

moved f&V cold aceLoue precipitation suspeuded in 90 ml of alcohol at room temperature; a fresh solution of 4.5 gm of sodirLm hydrosulfite in 30 ml of water was added, the flask was st,oppered, aud the mixture was shaker1 vigorotksly for 10 minutes. The sltspensiou of light yellow oil was diluted flIrther with water and ext,racted with :ibout 100 ml of petroleum ether, and the res\lltirlg weakly yellow organic phase was shaken thoroughly with Fjajo aqueous potassirlm hydroxide containing l-29$ sodium hydrosulfit,e. The PC’troleum ether lager was t.hen extracted with 50 ml of Claisen’s alkali (35 gm KOH in 25 ml water, diluted to 100 1111 with methanol) t.0 which :J ml of saturated aqueous hydrosulfte has been added. The alkaline layer was drawu off and plared ill a separatory funnel coutainiug 50 ml of ether, and two further rst.ractions were macle. The t ot;tI alkaline liquor was shakeu with the coveritlg layer of ether. The Fashed alkaline layer was then trausferret] into anot.her separat.ory fllrrnel ro~ltainillg .50 ml of diethJ.1 ether, and dilutetl with 2-R volumes of 2-4% aqueous hydroslllfite soll~tiou. The dilution results in liberation of the frpp hydraqllinone, and when the mist ore is sh:~kct~~. t.he quinol passes iut.0 t,he ether layer lo give :i pale yellowish sollIt ion. This was separated and wash(ld with X~IICOIIS sodium hydrosu1fit.e soIlLtioll all,1 then with water. Oxidation of the cIllillo1 t 0 the quinonr was ac~complishetl with silver oxide ;I gm/5 gm starting material). WIPI~ it x:1’: uttcessar?- to process larger smollllts of lipid, the (tua1Ltities of reagents risctl wrre ilrcare:xsed prol)orl ioIlbVi1.S

at+.

A ‘i-kg portion of fresh tissue n-as cut into sun111 strips for liornogenizati~~r~ in n ‘l\‘ariug Bleltd~,r. Blending was performed a~ thr high speed setting for 2 minutes to give :I 40% homogenate it1 distilled water. Thr homogenate was made 1 s with HCl. H,vdrolysis was carried c111t with rapid mechanical stirring at 75’ for 2.5 hours. The hydrolyzate was nrrltralized t,o pII 7.2 wit,h Sr10lmI. The resulting brown liquid was filtered throllgh :L large Biichner frmnel. The residue was washrtl rstensivel?; with water :tlld then lyophilizcd. The lyophilized residue WIS groc~rld w-ith a nlortar alit1 pestle and girldcd :I browu powder which was cxstractcd with chloroforlll-rnet,hallol (2:l). After t,hc lipid extract W:S obtained, the l)hospholipids were prpcipitatctl with cold ncelotle, after which Fieser’s (21) rctlllct ive isolat.iotl proccylllre was applied to the ant olle-soluble portion. spectra were recorded of thta lipid extract artd of the et,her extract ohtainecl at the completiou of thp rrdrlctiotl 1~1th brforcl :o~d nfier silvc,r oxide

51s

HAMILTON

AND DALLAM

cerned with the development, of a reliable vitamin I< bioassay. This was done so that various fractions of &sue extracts could be bioassayed for activity. The overall procedure was much the same as that “riginally used by Dam (22) and the workers who followed him, with the exception that soybean meal was substituted for the ether-extracted fish meal and that blood was obtained by direct heart puncture instead of venipuncture. Whole beef liver tissue was assayedfor ENZYMIC HYDHOL~SIS phylloquinone activity by supplementing A 2-kg portion of tissue was homogenized in a t.he diets of deficient, chicks with the lyomanner similar to that described for the acid philized tissue. In a typical experiment there hydrolysis with the exception that the blending n-as a control group, each member of which was performed in pH 5 acetate buffer rather than received 0.01 mg phylloquinone orally per &stilled water. The homogenate was then inday. A second control group n-as always cubated with 10 gm of crude fl-glucuronidase maintained on the deficient diet for the (Worthington Biochemical Corp., Freehold, New duration of the experiment, to check the Jersey) at 37” for 2 hours. The mixture was then abilit’y of the diet to produce and maintain brought to pH 7.2 with NaOB. The digest was then a vitamin Ii deficiency in the animals. There processed in the same nlanner as the acid hydrolywas also an experimental group n-hich resis, and both filtrate and dry residue were processed. ceived a supplement of the material to be tested. Several experiments were performed RESULTS AND DISCUSSION in which whole beef liver was used as the I;se of bioassay. In the period following diet supplement,. In all casesit \vas found the discovery of phylloquinone, many studies that, deficient animals, after receiving this were conducted OII plant material and a supplement for 2-3 days, began to lose the variety of bacterial species.Throughout the deficiency symptoms and their prothrombin work conducted on the isolation of phyllotimes returned to normal. Figure 1 shows quinone from both plant and bacterial the results of one such experiment. sources performed by many investigators These results imply that phylloquinone is over some25 years, the same basic technique present in beef liver. Although this evidence of isolation ha.ve been utilized. These same is indirect, it is reasonably wellfounded in techniques, however, when used to isolat’e view of n-hat is known regarding the specphylloquinone from animal tissues, have to ificity of phylloquinone and similar comdate been unsuccessful.In the middle 1950’s, pounds in alleviating elevated prothrombin shortly after 1Iartius’s proposal that phyllotimes. In addition, similar observations were quinone was involved in oxidative phos- made several years ago by Dam’s group (23)! phorylation, a great deal of work was done who used a somewhat different technique. to learn which one of the I< vitamins occurs Under the assumption that phylloquinone in animal tissue. In all of this work the ap- activity is present in beef liver, the next seriesof experiments attempt,ed to determine proach was basically the same; the vitamin was sought in the lipid extract, of the tissue whether this activity could he found in a by using column and paper chroma,tography total lipid extract obtained from lyophilized with subsequent spectroscopic analysis of beef liver. The extract was examined in vielr the various fractions. of the vitamin’s lipid solubilitv and of its The approach used in the present work demonst’rated occurrence in l&id extracts was similar to that, wed by previous workers, from plants and bacteria. The experiments in the same manner as bewith some modifications. Rather than be- were performed ginning with an attempted purification of fore except that the lipid supplement,swere lipid extract, the initial studies were con- given to t,he animals orally and separate oxidation. The yellow aqueous filtrate obtained from the acid hydrolysis was mixed with FeC13 (0.1 gm/lOO ml filtrate) on a mechanical stirrer for 1 hour. This was done to oxidize any free hydroquinone that may have been liberated into solution during the hydrolysis. This solut,ion was diluted with 2 volumes of water and extracted several times with 200-ml portions of petroleum ether. The combined petroleum et,her extract was dried by passing it through Na2S01, and the solvent was evaporated.

oI

4

12

H DAYS

1 (i

"0

ON DIET

FIG. 1. Effect of feeding lyophilized beef liver to vitamin K-defirient chicks. A, Coutrol; received vitamill K-free diet plus vitamin Ki (0.01 mg in 1 ml soybean oil). 0, Received vitamin K-free diet for entire experiment. 0, Received supplement of lpophiliaed Points were obtained by averaging the beef liver (.jOy6, deficient diet mixed with 50 yi, liver). values of the six members in each grorip. I)iet supplement, was st.arted WI day 1-l (see arrow’). (:r:tph is plotted selnilog~trithr~licnll~.

from t,hcir diet. Figure 2 shows data from one such experiment. It is seen that the lipid supplement caused the high prot hrombin time to r&urn to normal. This figure also show that. supplementation of the deficient diet with the tissue residue remaining after lipid extraction produces no change in the elevated protjhromhin time. This indicates

t)hat all of the phglloquinone act,ivit’y Ivas present in the lipid portion of the tissue. T,yophilized beef liver mit’ochondria and the total lipid extract from the mitochondria were next, examined fort heir effect, on vibmin Kdeficient chicks. Figure 3 shows a curve which is very similar to that seen in Figs. 1 and 2. (When t,he phospholipid fraction of

0

I

1

I

I

I

4

8

12

16

DAYS

ON

1 20

DIET

2. Effect of feeding total lipid extract from beef liver to vitamin K-deficient chicks. A, Control; received vitamin K-free diet. plus vitamin K,. 0, Received supplement. of total lipid extract of beef liver (approximat’ely 150 mg,/animal/day. 0, Received supplement of the residue remaining after lipid extraction of beef liver. Points were obtained by averaging the values of the six members in each group. Diet sttpplement was started on day 14 (see arrow). Graph is plotted semilogarithmically. FIG.

whole beef liver and liver mitochondria was examined for vitamin I< activity, none was detected.) Thus it was concluded that mitochondrial lipid cont’ains a compound which is capable of relieving vitamin Ii deficiency. This agrees with the results reported by Dam’s group in 1956 (23). Their report indicated t,hat phylloquinone activity was

contained primarily in the mitochondrial fract,ion of beef liver, although the nuclear fraction appeared to have some activity. Because of the isolation procedures used, it was later suggested that the values obt’ained for the nuclear fraction were probably high. The present) result,s would also support t,he work of AIsrtius (21), who presented evi-

I

1

1

8

DAYS

I

1% OX

I

4

16

20

DIET

FIG. :3. Effect of feeding beef liver mitochondria and mitochondrial lipid to vitamin Bcleficient chicks. A, Control; received vitamin K-free diet plus vitamin K,. 0, Received sllpplemeut of lyophilized beef liver mitorhondria. 0, Received supplement of total mitochondrial lipid extract, from beef liver. Points in cont,rol group were obtained by averaging rhe values of the forw members in that group. Poiuts in experimental groups were obtained by averaging the vallles of the eight members in each grorlp. Diet supplement started on day 12 (see arrow). Graph is plotted semilogarithmically.

dence I\-hich indicated t#hat, menaquinone-4 was in the liver mitochondria of a number of animals. This conclusion was reached not on the basis of a bioassay but rather on results obtained by using countercurrent distribution. He reported a similarity between the 1)artition coefficients of menaquinone-4 and the radioactive material obtained from lipid wtmcts of liver mitochondria isolated from

various animals which had received labeled precursors. The vitamin was not isolated and has not been examined by definitive chemical and physical st,udies. In the present work the mitochondrial lipid extract was separated into several fractions and was assayed for activity to guide isolation of the vitamin. [Because of the large number of chicks in each group to re-

ceive the various fractions, it was necessary to begin with :I large amount, of total lipid extract! which was resolved on a column of sufficient size (8.9 X 152 cm) to accommodate several pounds of the Decalso absorbent.] It required several days to resolve t,he amount, of lipid that would t,heoretically contain enough pylloquinone to bc detcctable by the bioassay procedure. This was recognized as a great disadvantage since it had been sho\~n previously in several laboratories that prolonged exposure of phylloquinone to certain adsorbcnts used in purification caused dccomposit,ion of the vitamin. Forty gm of beef liver mitochondrial lipid \verc resolved into four fractions by column chromatography on Decalso. The eluting solvent and amount of material obtained from each fraction were (a) 5 liters isooctanc, 2 gm; (b) 4 liters of 5 ‘Z ether in isooct,ane (v/v), 4 gm; (c) 3 liters of 10 7; ether in isooctance (v,/v), 6 gm; and (d) 5 liters of methanol, 15 gm. The collectjed fract’ions were processed and the oil was given to deficient chicks. (I’hylloquinone and ubiquinone-like materials predictably would be present in fraction 2.) The total unresolved extract was able to reduce the prothrombin tjimc of the deficient animals (see Fig. 3), but none of the fractions showed any activity. Akhough it was felt that such a fractionation of the total lipid would he quite valuable, further attempts were discontinued.

permit detection of any vitamin I< present n-ascalculated. Small columns (2.5 X 40 cm) were used with eit,her Decalso or silicic acid as adsorbents. Before chromatography of the experimental material, several pilot studies with authentic phylloquinone were made. In these studies ltnown detectable quantities of either phylloquinone or menaquinone-4 were added to a portion of lipid extract, and the sample was chromatographed. Previously developed solvent sy&ems were used to elute the column, and recovery of the added phylloquinone \vas calculated from spectral analysis of the various fractions. In all cases the recovery exceeded 90%. These pilot studies demonstrated that small amounts of added phylloquinone could be separated from the &her lipid materials and could be detected spectroscopically. We assumedthat, any naturally occurring phylloquinonc would bcain a form similar to the phylloquinono used in the pilot st,udies,and it was therefore felt that such methods would be reliable for its detection. When the two adsorbcnts and three different solvent systems, lvhich gave good separat’ion in the pilot studies, were used, the lipid extract from beef mitochorldria was chromatographed. 111 a typical cxpcrimcnt 5 gm of lipid n-as dissolved in petroleum ether and applied to a silicic acid column. This amount of lipid could contain approximately 370 pg of ph~.lloquinotle, if all of the vitamin \vere onl\~ in mitochondrial lipid. 111 all these experiments, spectral Ch.emical and ph~ysicd rxawz’nafion oj’ w itoanalysis of the eluents indicated that no chondiial lipirl. Since the previous results phylloquinonc L\-aspresent. Therefow, the indicated that phylloquinone was present, in resulting fractions were concentrated to apthe lipid extracted from liver mitochondria, proximateI>- 0.2 ml for rechromatography on methods were examined that would lend thin-layer plates. Authent,ic phylloquinone, themselves to the isolation of t,he vitamin mcnaquinonc-4, and ubiquinone-10 were from such a tot,al lipid cxkact. The only chromatographed as standards along with purification of the mitochondrial lipid pe- the three unl~~~ownfractions. Three wlvent troleum ether extract prior t,o application of systems were used which were known to sepat,he lipid to :I chromnt,ography column \vas rate the vitamin I< from ubiquinonc and the precipitation of the phospholipid fraction from lipid material present in mitochondria. by 4 volumes of cold acet,one.Several chroma- After development of the plates the only t,ographic procedures were used to detect spot that could be identified was that, of any vitamin 1\ in the acetone-soluble fmc- ubiquinone-10. This same procedure was tions. By using the value arrived at by Dam follo\ved \\-ith the c,luentsobtained from both for the concentration of phylloquinone in the Decnlso and silicic acid columns. 111both liver, 0.01 pmole/gm of tissue (23) as an ccws several experiments produced negaapproximation, the amount of lipid neces- tive results. Spots 011the thirl-layer plates sary to be applied to :I colllmn which ~~kl and paper strips uwc detected in the dark

\vith :m ultraviolet light. The sensitivity of this met hod v;wics some\\-hat hut has :I lower limit of :yqmmin-mtely lo-15 pg of cluillolw. The platw wre next sprayed \vith neotetrnxolium chloride, which, under controllecl coiitlitiow, lins a lower limit of detectiw of 5 pg per cluiwne. The failure to dctrct any. spots ditl not conclusively illdicatc tlw at)wiice of l)h\~lloclui~~oile in the unl;iiou-11 mt cri:rl ; mthcr it iiidicatcd th:rt if the vitamin \\xs prwe~~t, it \v:I,~ t hew ill clu:uititiw belo\\the limit of detection. Therefore, the thilll:r\w pl:tt (+ \vcre rldctl into L’-cm squaws in the :~rc:w ~herc I)li).lloc~uilroll~~ \~ould hc fount1 :w Iwetlictcd t,y the iiliglatioli of the st atitl:rrcls. I~;iglrtecw of the Scni s~~u:irc :mw \\wc wxpecl from 11~ pl:ttc iirto sqxuntc

220

'4 0

tutxs, and the material was shaken vigorously with chloroform to extract any organic nlatcrial that, ww present’. After centrifugation to rcn1ove the silica gel G, the chloroform IV:P cvqomtt~d and the residue was dissolved in c\-clohesane for spect ml esnrninution. The louver limit of detection of authentic vitmlin li iI1 tlic: ~pcctrol)hotomcter is 3 pg. Konc of the cornhined nwtcri:d tlisplt~yed the chnrwtcristic spectrum of ~~liylloq~iir~orle. ICigurc 1 (curve C’) shm-s the qmtrum of 1hc fraction, following column md thinI:\\-er chrOi11:Ltoglnl)ll!-, ill which phylloquirmlle would tx found. Curve ‘4 shows the spcctrllni of :L s:miplc of :rnthentic phyllocluinoiie. .Iftcir phyllocpinorlc mm added to 5 gin of niitocho~idri:d lipid nntl chronmto-

260

280

3 00

320

34 0

graphed, fraction 2 was collected, conceittrated, and chromatographed on thin-layer plates to obtain the added phylloquinone. Curve B shows the spectrum obtained from the isolated phylloquinone. This experiment was used to calculate the percentage recoverv of phylloquinonc from the procedure, which was 92 ‘7. The results obtained from cxaminat,ion of the mit,ochondrial lipid extract are in agreement with earlier n-ork reported by Green and Lester (35), Redfearn and I’umphrey (26), and Crane ( 2i), all of whom reported unsuccessful attempts to demonstrate the presence of phylloquinone in mitochondrial lipid extracts by spectral a,nalysis. Chemical and physical e.camination of liver lipid. Since repeated attempts to demon-

strate spectrally the presence of phyllotquinone in the mitochondriul lipid consist’ently produced negnt,ive results, it was decided to reinvestigat,e t)he total lipid estract since the procedures used to isolate mitochondria in the required large amounts were very time-consuming. (To obtain enough beef liver lipid for analysis required approximately 7 days, but to accumulate the same amount of mitochondrial lipid, about 3 lveeks lvere required.) This decision was also prompted by an earlier observation that such a total lipid extract did relieve the deficiency induced in chicks by a vitamin I
(by weight) of unwarned lipid material is removed in the early steps of the procedure. Ahhough this procedure has been used by many workers in the isolation of phylloquinone from plant, material, it, was felt that the validity of the technique should be verified by using beef liver lipid. I\no\\-n amounts of synthetic phylloquinone or menaquinone4 were added to a portion of total lipid extract, and this mixt,ure n-as subjected to the reduction-purification procedure. One-bun dred, ZOO-,and 300-pg quantities of phylloquinone were added respectively to three 10.gm portions of beef liver lipid extract,. The et,hcr-extractable material obt,ained from the final steps of the procedure n-as oxidized with silver oxide and examined spectrally. In all three casesa typical phJ.lloquinone spectrum was obtained. Slight, impurities were present’, but these were separated from the vitamin by thin-la!-er chromatography. Further investigat,ion rcvenled that, when as little as 50 pg of phylloquinone n’as added to a 10-g-mportion of the lipid extract, the presence of the vitamin in the final ether extract was easily determined spectrally~. (Recovery in the latter caw was calculated to be 5s 9:. When a higher concentration of vitamin I\: was added to the total lipid, recovery was usually in excessof S5 ‘;I.) Thus it was estimated that the lolver limit of the method approached the 50.pg level. Thus, even if the overall procedure TvaRonly 50 “5 efficient, it Ivas calculated that there would be enough vitamin present for detection either spectroscopically or chromatographically. In a continuation of working with the beef liver extracts, from which the phospholipids had been removed, attempts were made to demonstrate the presence of phylloquinone by using this procedure. Seven experiments utilizing this technique all produced negative results. In all of these experiments spect)ra I\-ere recorded on the final ether extract) both before and after silver oxide oxidation. In addition, each of the extract)s were concentrat’ed to approximately 0.1 ml for thin-layer chromatography but revealed no phylloquinonc. Acid and enxymic hydrolysis. It has been reported by Hoskin et al. (2s) t,hnt when menadione is administered intramuscularly

ISOLATTON

()F

\-ITAMIN

to rats, two urinary excretion product,s are menadioue diglucuronide and menadione monosulfate. Experiments were therefore dwigned to study t,he possible occurrence of phylloc~uinont~ in the liver in such a cottjug:ttc~l hvdroquinone form. It was felt that if phylloqktlone occurs in t,he liver as a glucurot~ide or sulfate, then the work conducted thus far on lipid extracts would have indeed produced negative results (altho~tgh the a(lucous phase obtained from such extracts had bwt1 preliminarily investigated for its quittonc~ content). Hoskin’s group (28) had demot 1st rat cd that the menadione glucuronidc and menadione sulfate could be hydrolyzcd \vith dilut,e HCl. In addition, the glucuronidc \\-a~ susceptablc to the action of &ghtcuronidxse. It was felt that similar cotltiitiotts I\-ould be equally effective on glucurotiidw or sulfates of phylloquinone or Cta.tnin Ii,. After chemical and enzymic hydrol~~w wrc performed, the ether ext rnct ot the queous filtrates, \vhich could contain either hydroquitione or quinone, was :m:~lyzed spectroscopically and chromatogr:rphicall\-. So ultraviolet-absorbing materials wre foutld, and thitl-layer chromatogr:yh~- of the material revealed no spots I\-ith I< I‘ values similar to phylloyuitione. The rcsi(luc~ from each hydrolysis \vns lyophilizc~d and ground to a powder, and t#he lipid MYI’-:cxxtract’ed with the standard chloroform-tn(,th,ztlol mixture. The resulting lipid cst r:wt s were processed according to 1;ieser’s redt:cti~e-isolnt,iott procedure. Spectral and chrom;rtogr:lI)hic analysis of the material showed no phylloquinone. lhxruir~afim 0s licer of othet~ar~ir~mlts. A\1though 1)iological activity in the chick assay n-w observed I\-ith the total lipid estract front l)eef liver and for the lipid extract obf:litwd from beef liver mitochondria, no physical widewe could be found for the pr~wnce of phylloquinone in such extracts wh~1 ~t:tnd:wd terhniqucs and some modified onw MXW used. Thus it must bc COW chided that the vitamin occurs in this tissue at :I concentration below the limit of dctectioti by the procedures emplo;\.cd or that it occurs it) some other form. Thcb wrly literature indicates the a~)p:lrctlt coticrtitrntioti of phylloquittone in the

Ii

Fl:OhI

T,I\‘IC

I:

Xi

livers of different speciesof animals to var!-. This variation was indicated by the ohserwtion that different amounts of lipid extract8 obtained from the livers of various animals were required to overcome the deficienq symptoms in the chick assay. Throughout the majority of the present work, bovine liver \vas used as a source for lipid. After unsuccessfully at tempting to demonstrate the presence of the vit)amin in bovine liver using :I variety of sensitive physicochemical techniques, it w,s decided to examine lipid extracts from livers of other animals. Total lipid extracts from rabbit, adult chicken, and swine liver \\-ere prepared. Two and three-tenths kg of each tissue n-as lyophilized, and the total lipid extract obtained from each tissue, after precipitation of the phospholipid with cold acetone, was sttbjetted to Fiescr’s reductive-isolation procedure. The final ether extract’ resulting from this procedure n-as examined spectroscopically, after silver oxide oxidation. The spectra gave no indication of phylloquinone. Each of the samples n-as chromat ogrnphed 011t hit1la!-er plates and developed as described befort. After development, the plates were sprayed with the dye, and t\\-o positive spots were observed from all three prepurations. Oiic~of these spots migrnttd toward the solvent front wry close to ph?-llocltlittottt~. The second spot moved only :t short disrattcc from the origin. k~nch of these spots W;IS scraped front the plate and eluted \vith chloroform for spectroscopic :utnlysis. In each case (rabbit , :ldu11-chicken, and s-;\vinc) the front -running spot displa!-cd the typical spectrum of the family of li \-itamins \vith absorption maxima at ‘269, *LAO,“4S, and 243 nip. (The conct~ntration of this materia.1 ~v:is veq. low., and it \\-:ksnecessnry to cornbine the front-rutitiiiig sl)ots front xcvcral scparatc chromatopln~es to obtain a spectrum \vhich clearly showed the four sharp peaks at the nhovc-tuentiotled n-:tr-c~letlg::ths.) The curve obtained front the chicken liver extracts is sho~~tiin Fig. .i, curve A1. This spectrum and that of phylloquit~onc, shown in Fig. 4, cur\-f‘ -1, are identical. Sitnilal spectra were obtained nfter cliroi~~:~togr:~~~li~of the rabbit and s\\-ine liver wtlncts. Alor+

526

HAhlILTOK

ASl)

DALLAM

1.0

0.9 0.8

220

240 WAVELENGTH

FIG. 3. Ultraviolet chicken liver. Curne potassium borohydride.

260

280

300

320

340

- MILLIMICRONS

absorpt,ion spectrum of the suspended vitamin K isolated from -4: Compolmd as isolat,ed. Curve B: One minute after the addition of Curve B was obtained with absolute ethanol as solvent.

over, these compounds, ~-hen mixed with a 1% solution of potassium borohydride in ethanol, showed the characteristic reduced spectrum typical of the I\ vit)amins lvith a maximum at, 244 rnp, as shown in curve R of Fig. 5. E’or comparison, the spectrum of reduced menaquinone-4 is shown in Fig. 6, curve R. The reduced compounds were easily converted to the oxidized form b>- silvcl oxide oxidation or by allowing t)he m:lterial to stand in air for some time. In each of the three samples, the concelltration of the compound obtained from 2.3 kg, wet weight,, of starting material was calculated by assuming a molar extinction coefficient) of 19,SOO at 243 rnp, which is typical for the I< vitamins. Rabbit liver had the highest concentration 5 pg/l gm of lipid ext,ract, and chicken liver had 3.33 fig/‘1 gm lipid extract. The concentration in wine liver ~V:LS not calculated hecause it was not

pure. (Estimat,es from spectral assays indicated that the total amount of compound in swine extract was far below that, in the chicken and rabbit preparations.) The isolated compound from each of the three liver preparations was rechromatographed on thin-layer plates along \\-ith authentic phvlloquinone and menaquinone4 as standards. It was consistently ohacrvrd that’ this compound moved with XI RF that, fell between phylloquinone and mcnnquinone-4 The same results were found by using two difierent solvent systems. Tahlc I lists t,he chromatographic properties of this compound which lwre identical in each of the three liver preparations, along \vith those of the known I\ vitamins. In addition, when several micrograms of the compound obtained from chicken liver UYW mixed with either phylloquinonc OI menaqunone-4 and chromatographed on thin-

1.0

0.9

0.8

0.7

> E-

0.6

55‘ Q

0. 5

2 E g

0.4

C

0. 3 0. 2

0.1

WAVELEXGTH

- XIILLIMICRONS

FIG. 6. Ultraviolet ahsorption spectrrlm of oxidized and redllrecl Inrllnqr~inol~c-~. (‘I~IW .I : Oxidized form. Cuwe U: Redwed fornl. Redllced specatrlml was obtained by mixing 1’; potassilml I-wrohydride wit,h a sollltion of mell:~qrGllone-i in ethanol. Spectrum was recorded 1 minlite later.

layer plates, two spots xvcrc alw:~~s found, and the measured ZZF v:~lucs wre the same as those found when the compounds I\-erc chromntogr:Lphcd separately. Xlthough this diffcwnw in RF v:~luc:: betwzn the unkno\~n 2nd phylloc~uinone \\-as small, rcycatccl chronxttogxphy demonstrated this diffcrencc to 1x1 consktent. The ultrnviolctj absorption spectrum of this con~pour~d \vould indicate that t he compound is :L 1,4-11:~l)hthoquinolle \vith some suhstitucnt in the 2 and 3 positions. I’urthcrnmorc, the spectral ch:tngcs brought :ll)out b\v thr addition of potassium horohyclridf arc indicative of such n cjriinoid structure. Since only microgram quantities of this wmpound UWE isolated, it, has notS yet been possible to pursue further ~h:~r:Lctcrizntioll l)!~ infrared and elemental annlysis, and

I~iological assq-. 13;rsed on the> cGtlcncc accumul:~ted and the literature Icports pcrtniiling to tlic isolation of pli~lloc~uitioiit~, it ~\ppcars re:won:kk~ to concludes that the isolnted compound is :m “:lnim:~l vitaniitk Ii.” The gross structlw of this comporn~d must tw idcnticnl to that of the kiw\\7i I\ vitamins, but cliff~wnccs iri fiw strlwi urc ma!- h(x irlfcrred from the chrom:~to~l~a.~~hic twh:Lvior of the conipouiid. The ~lirom:~togxpliic bclinvior of the compou~lcl indiwtw that it is not idc~liticnl to c+ther phyllocluillotlc or metincli:illoiie-4. It nio\-(‘8 with an RF close to that of ~)h~llocllli~rol~c.;\I:wtirls (16) has rcportcd that the vitamin T
52s

HAMILTON

matter for speculation, the differences in chromatographic behavior between the two standards and the newly isolated compound are undoubtedly due to slight differences in struct)ure, probably in the subst,it,ut.ed side chain. The possibility t,hat, the difference may be due to A side-chain length, that, is, 5-, lo-, or 15.carbon atoms, is not indicated since compounds with t)hese side chains all move more slowly, i.e., have smaller RF values than that of the isolated material. It, is possible that the isolated compound may have a L’O-carbon isoprenoid side chain with partial unsaturation. The isolated compound ma? not be considered to be of the menadione type because compounds without a substituent in the 3 position either do not’ exhibit an ultraviolet, absorption band in the 270 rn/* region or show a shift in t,his band toward the lower wavelengths. Furthermore, compounds of this type have lower RF values than phylloquinone or menaquinone-4 in all chromatographic systems used. The possibility that this compound is one of the ubiquinonc series can also be eliminated on the basis of its ultraviolet spectrum and chromatographic properties. Recently, Lest,cr and White (29) have isolated from bacteria a menaquinone-4 jvhich lacks a met,hyl group in the 2 position of the quinone ring. The ultraviolet spectrum of desmethyl .I<, (20) indicntcs that the compound n-e have isolated from rabbit, chicken, and swine liver is not a desmethyl. The structure of t,he compound isolated from swine, rabbit,, and chicken livers is tentntively proposed to he:

.4X>

DALLARI

slightly lower than phylloquinone, the above structure is proposed since it, would be predicted to possess the same chemical and exhibit the same physical properties as the isolated compourld. The exact, location of the double bonds in the side chain is open to question. The second compound obtained from each of the three liver preparations appeared to be present in greater amounts t,han the first, compound. This was estimated by visual examination of chromat,ograms sprayed with the dye and compared with standards. This compound moved only a few millimeters from the origin in all of the solvent systems used in the thin-layer chromatographic systems. The ultraviolet absorption spectrum of the compound, as obtained from rabbit liver, is shown in Fig. 7, curve A. Curve B illustrates its spectrum obtained after reduction with 1% potassium borohydride solution. The oxidized form shows absorption maxima at 281 and 265 mh. Identical spect’ra lvere obt)ained for chicken and wine prepar:ltions. This oxidation-reduction react,ion \\-a~ also rcversiblc with either air or silver oxide as oxidants. The second compound gave a negative Dam-Karrer reaction, and the ultraviolet spect,rum is not similar to any of the ubiquinone series of compounds but is ident,ical to that of a-taco-phcrJ.lquinone (31, 32). Its RF values do resemble some of the ubiquinone homologs. Sollocnsa and Crane (30) recently report,ed t,he isolation of several compounds capable of undcrgoing reversible oxidation and redu&on from

~,CH=~-C&CH~CH=~-~H~~H*-~-~H~~H*~H~-~-~H~ !I P-Methyl,

!I

3-A S-dehydrophytyl-1,4-naphthoquinone

Because the isolated material showed (a) in the oxidized form, four charact,eristic peaks in the ultraviolet region; (b) in the reduced form, one characteristic peak in t)he ult)raviolet region ; (c) had an RF value that \\-ould 1,4be given only by a 2,%ubst,ituted napht hoquinonc with four isoprenoid units at position 3; and (tl) had an RF value onlg

lipid extracts obt,ained from beef heart mitochondrin. One of the compounds reported behaved chromatographically very similarly to the one described here. The spectrum of their compound was not t,he same, however, as the spectrum shown in 1Jig. 7, curve A. Crane (30) has suggested that their compound may be a benzoquin-

dation, during the reduction a-tocopherol (31, X2).

none. It, is felt that, the compouud obtained in this work is a benzoquinone because of its chromatogmphic behavior and spectr:~l chnrxteristics, and that it is probably a-tocoI)herolquinone Ivhich resulted from the osi-

for

Thr allthors his espcrt.

procedure,

arc grateful to i\Ir. assistance throughorct

of

John RI. llouk the project.

.-I~/lrlent/~~~/. :I recent article of .\IatschincI ef al. [Hioche~~isli~~ 6, 12U ( 19BT) reported the detection of :L mythoyuinone isolated

from raw beef liver that showed vitamin Ii

O.GG

O.Kl 0. 63

0.53

O.(i5 0.G 0. G(i 0 .3!)

0 .(i4

0.63 0.64 0.37 “ Solvent ethrr iv/v); were coated

1.0

act,ivity in the chick bioassay xd \\.hich appeared similar spcctric:~lly to the group of 1\ vitamins. This group also reported that rabbit and pig liver vit:rmin I\ wtivitics v~~c diminished by cooking. These results :md

A4, 5’34 diethyl ether ill solvent B, bcllzene. Thklayr with silica gel (:.

those reported h;\. us point out that \vhcn dealing \vitli compounds from different tissues, special cousidcr:rtion must be givcw to each tissrle. Tissues from different species possess inherent differences in rhemical cornpwitiou, :u~d these tliffcreIlccs ni:~~- 11c m:~g$-

petroleum plates

-

0. $1 -

0.8

-

0 .i

-

22

0.4 -

-3 4

0.3 0.2 0.1

-

I

I 220

I

I

240 WAVELENGTH

FIG. 7. Ultraviolet absorption spectrum liver. C?~roe A: Compound as isolated. Curve (‘urve B was obtained with absolute ethanol

I

I

260

I

1

I

‘80

I

I

300

III

320

- SIILLIRIICROK;S

of the second compolmd isolated B: After the addition of potassilun as solvcut.

from chicken borohydride

340

nified by factors such as diet. Any of these differences might atiect the solubility and stability of vitamin I\. Alcohol-chloroform extracts of beef, rabbit, swine, and chicken liver, when subjected to the reductive isolation procedure by us, behaved differentl\* in that no vitamin I\: n-as detected in the purified extract from beef liver. Since vitamin K n-as detected is t,he rabbit, swine, and chicken purified extracts, we conclude that some lipid material interfered with the procedure \\-hen applied to beef liver extracts. The loss of vitamin li activity, reported by the above authors, in cooked rabbit and pig liver and not in beef liver suggests either the presence of :t compound(s) in the tissue of tht: t\vo former species lvhich when heated led to the dest,ruction of vitamin I\;, or the presence in beef 1ivc.r of a compound which protected the vitamin from heat destruction.

I'.,

12.

2.

~ALLAM,

c.,

BiophJ/s.

.\ND

.lcla 1:.

NI'IWLITZO\\-,

I).,

13.

.\ND

~~NDIMOS,

\$'.

\v.,

&AU 25, 439 (1957). J. P., .\NI) SL.\TEIX, E. C., Uiochim. Biophys. -4cta 27, 122 (1958). 4. BIwDIE, -1, F., ANI) B.\LL.\NTISE, J., J. Viol. Chen,. 235, 232 (1NiO). 5. DAM, H., Biochem. J. 29, 12i3 (1935). 6. DAM, II., SCH@NIIEYDEI~, F., .\ND T.iGEHASSEN, E., Biochem. J. 30, 1075 ilYl(i). 'i. &ma, ;1. J., AND COLLENTIXE, c;. E., A?u. J. Physiol. 164, 716 (1951). 8. BRODIE, A, I?., :lDslr. 13fnd .lleeliny Am. Chem. sot. p. 52 (1957). WEBEK, RI. W., .\ND (+Ii.\Y, 9. ~ROI)IE, -1. F., C. T.. Biochem. Biophys. ~1~1~25, 448 (195i). 10. BRODIE, A. F., .\SD B.\LL.IWINE, J., J. Viol. Chem. 235, 226 (1960). 11. L)AM, II., GEIGER, A., GLAVIND, J., K.\RRER, Biochi~~.

3.

Co~l,.~-Hoos~~~t.~,

ISLEI{, I,.

W.,

~
BINKLEY,

C.,

II.,

RI-EGG,

IT.,

WINTERWEIN,

E.,

.1N,)

22,310 (1939). S. B., THATER,

L>. w.,

:1ND

L)OISY,

131, 327 (1939). CH~P:~I~D-DI,I~-JE.\S, A.,

.IND

WISS,

O.,

Hell:.

14. 15. lfi. Ii.

18.

19.

20. 21.

BioChiVl.

13, 152 (1954). u.,.,

11.

H&.

.4., ~~.\~COR~ITOI).\LE, E. A., J. Biol. Chem.

'rz. j&ItTILTS,

MCKEE,

\v., II.,

s.

22. 1.

~UlItEl~,

tii.\LO1MON,

24. 25.

fiiophya.

2G.

C’hinr. .Icfa 41, 7% (1958). NOLL, H., J. Hiol. Chewz. 232, 919 (1958). BISHOP, L>. II. L., I’.\NI>T.\, K. P., AX,) KING, II. K., lliochem. J. 83, 606 (1962). Mwur-s, C., .\su li:ss*:~t, II. O., Hiochem. %. 331, 1 (1958). (JUICK, A. J., Am. d. I’hysiol. 140, 212 (1943). SEWERS, IT. II., “l’rothromhir~,” p. 500. Harvard Univ. Press, Cambridge, Mass. (lN2). SF.HI., E., Clw,~. 2. 82, 323 (1958). LESTER, R. L., ANI) I~.\MAs.~I~M.\, T., J. 13iol. Chem. 234, 072 (1958). E'IESER, L. F., J. AWL. Chew. Sot. 61, 3467 (1939). I).Liv, Ii., Salure 133, 909 (1934). (;I
A-inn.

E.

It.,

ASD

~'C.\IPHILEY,

A.

M.,

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