2 2

BBA 55234

B1OCHIMICA ET BIOPHYSICA ACTA

P H Y T O L METABOLISM IN T H E B O V I N E

STL'A RT PATT~ iN .xr¢l) 3.. A. P,F NSON Ltpids Lah,.'atory, "l'tw l 'e~.s) 'h 'a, a Slate I';;irrrs;; , l',m'crsiev Park, 1'.. (I ' .S. .4.) , aml Depart. merit ,~f Marine Hiology, Scr,pp: J,l~tittaion ,~: o:~'ci~t,,grapM', ~'ni,'~z~srtv o! Cah l,,rma, La Jolla. CaliL (U,N.,-l.)

(I,~cccivcd I)cccmbcr z7th, ~9~'5)

sUMMARV

~. Phytol, dilaydrophytol and phytanic a d d were identified in ;u lounts I,el,~w I°0 of tile total lipids in runlen c . n t e n t s from twt, normal lactat ing l tolstein cows. The plasma lewq of phy tana te n'~easllred in ,)he ~tt lhest, animals was of tilt. s;tnle magni tude as tha t tound in tile tureen and tha t reported in milk fat.

2. A t racer experiment with uni formh' ~4C-laheh'd phytol indicated conversion to d ihydrophytol and phytanl, ar id in the runlen. (mh" [rec phytol, as .pt .~sed to tha t ,n chlorophyll, appeared to be hydrogenated.

3. The relationship of lh(,se findings to high h.vels .ff 1)hytanic acid detected o|1 oeca,;i ns in c~)w blood lil)i(ls and to the hui/lan ;tflticti.n knowrl i t ' , | , ~c lS l t l n l ' s

syndrome is discussed. 4, Thin-layer and gas-rhromatogra l>hic tv(hni t lur , u~('tul in th(, i . . l a t ion ,

identification and analysis of I~hy'toI and ,~omr ot it- dcrivativr~, werr drvehq~e(I.

1 N~I.~OII L'CTION

Phytol (3, 7, I I , IS-tetramethyl-e-hexadr, 'cn-l~i ,I ) i., , on ta in rd in thr c h l , m . ],hyll molecule 1) 3" ester linkage and rq)re~ents aiqm~ximately om,-third .1 tht, nla.s 4~1 both ehlortqfltylls a and b. M:nnmalian metal)olisnl o[ phy t . l has a t t rac ted a t ten t ion recently because of the c(,mpound* po>sible role in Refsum' .~ di~caseL a rare nru- rological disorder in hmnan~ characlerized b~ very high levels ~f phytanic acid (3, 7, I z, I5 - te t ramethyl hex'ldecanoit .acid) i . the blood serum and -r:. 'an lipide. ~. Phv t an i r acid has been identitied also as a t ra re coqst i tucnt of milk fat:' ~ and in bovinr blood~, r Exogenous phytol appears to he a ', '~Ul-t " Ill phytanic acid in Rel~.tnn'~, disr.t~.e and an inh,,ln inadequacy fi~r oJ-oxidalion may account I . r accumulat ion of this a,'id ~.".

Mammalian metabolism has been confronted with phytol for a hmg I~:riod of evolution. The remarkable nml t ibranchcd s t ructure ot it and of its closely related derivative, phytanic acid, suggest the nred for be t te r umters tanding of phytol meta- bolism. The bovine appeared part icularly valuable for such studies since it consumes substant ia l amounts of phytol and prod,aces human foods (meat and milk products) ,

13iochim. Ei,,phrs..-tcta, l.:' 5 0960) z2-3z

PHYTOL METABOLISM IN THE BOVINE 23

comaining phytanic acid. Moreover, in extensive studies of bovine milk and brood lipids (e.g. refs. 9 and zo), we have never seen any indications of the high levels of phytanic acid reported by DUNCAN AND GAR'rO~ 6. Thus the possible existence of abnormal as well as normal synthesis of phytanie acid in the bovine suggested the need for rematch.

MATERIALS AND METHODS

Plo'tol and its derivatives Phytol of better than 9C:°o purity by gas-chromatogt'aphic analysis was ob-

tained from both Mann Research Laboratories and Calbiochem. Corp. Dihydrophy- tol (3, 7, zI, xs-tetramethylhe~adec~nol ) was produced from phytol in essentially quantitative yields by conventional hydrogenation. Phytanic acid was made from dihydrophytol by the method of KARRER, EPPRECHT AND K6NIG tl. Methyl phytanate was prepared by refluxing the acid in x% (v/v) HsSO4 in methanol. This ester after purification by silicic acid column chromatography remained a liquid at 2 °. Details of these syntheses are presented elsewhere t,.

Trimethylsilyl ethers of phvtol, dihydrophytol and other xeference alcohol. were made in pyridine essentially as described for hydroxysteroids 13.

Unifiwmly t'C-labeled phytol was prepared from a 3-day incubation ~f algae (Chlorella pyrenoidosa) with 5 mC of ~*CO2. The lipids, extracted from the ~et cells with hot ethanol and pyridine, were salxmified, the unsaponifiable matter was sei'a- rated on a preparative thin-layer chromatographic plate and the phytol~raction recovered by extraction with ethyl ether of the silica gel scraped from the appropriate area of the plate. The radioactivity in this extract co-chromatographed with phytol both by gas and thin-layer chromatography (Figs. I and 2).

l ! = [ t

: ' I 1 1 1 r l "

i i . . . . . . .

1 4 Fig. z . ( ; a ~ ; c h r o m a t o g r a m ot p h y t o l (smooth lint') a n d its C ac t iv i t y ( ragged fine}.

l'repamtiolt of !ipid.~ Sample~ of rumen ingesta, 5o-z5 o ml, were recovered from milking Holstein

cows by means ~f a stomacll tube. These animals were on a normal ration of mixed hay (alfalfa and grass), corn silage and grain. The freshly drawn material was imme- diately combined with an equal volume of absolute ethanol. This mixture was adjus- ted to pH 2 with 5 M HCI and allowed to stand 2-3 h after which ~t was filtered through ~vera l thicknesses of cheese-cloth to remove coarse feed particles. These particles

Biochim. Biophys. Aaa, t25 {z966) 22-32

~4 S, P.4,TTON, A. A, BENSON

were xvashed wi th two more volumes t)f t,oi:ing ethanol which were allowed to drain int() the original filtrate. The total fi l trate ~v~ ; centrifuged at IOOO rev,/rain for zo rain to remove suspended matter . The clear s cpe r na t an t was decanted. The sediment and feed particles were rinsed with small ix)rtions of e thyl e ther and these rinsings added ~o the supernatant . This ext rac t then was reduced to dryne.~s at 4 °° with a ro ta t ing evapora tor under wate r -pump vacuum. Lipids in the residue were defined as those substances per IOO inl of original ingesta tha t wnuid d i ss .h ' e in 2o ml of chlor(ff(~rm- methanol (z : z, v,~v).

~ . " ? ~ . ¢ •

' ' t • ' • ' " "

, , , ~ I I ~ I A A B B C 0 A B C D E

Fig . . ' . Th!tl-iqLver chr(mlatogram (light bat'kgr(rafM) alhl auluradi(Jgram dcrlxcd lrom it (d;irk backl~romld). \ pt,vtol dihydrophyt(~l a n , l ~t" ph>tol. IL [~(" phvl(~l. U, phylol Ii. ,lihxdr~q~hv- h)l. Silica ~e|-ARNit:~ o l d chlor()hwm oct'tic arid i re ' l : ~. v # ) .

]:ig. 3. "l'hill-lay~,r Cl/IFOllHltOgr;lltl t)[ II]l'lhv] ~'51t'I'.% lie Jill[IV acids A, plillllltitlt., 1~. Mt.arMt.. (',~dcat¢.. >, cd~'atc alld phvtana lc . 1,;, phvtanatc , Si l i ta gt'l-lJ ilil~[ light pctroh.unl ethyl cthcr !1t(15:5. x ~ ) .

I1] the interest of detcrmining tht ~ lwesence ,>f pliyh)l and related t '(nnlmunds in cows blood, a 4o-nil bh~od sample wa~ taken at the same time as one <)t the runwn ingesta samples and the lipids extracted essentially as described l,w the rl.lllltql sampl,,s but without pH adjustment .

Saponification of lipids was accomplished in x. 4 M KOH in e thanol at reflux for 3o rain. Unsaponifiables were recovered from the reacti~m by dilut ing it with an equal wflume of water and then ext rac t ing several t imes with redistilled hexane.

lli.chim, l¢ioph_vs..'tcta. I z 5 (190~,) -'z-32

I'HYTOL 3IETABOLISM 1N file BOVINE 25

The soaps were decomposed in the extra~'ted solution by warming with an excess ~t IM HCI and the resulting fatty acids were obudned by hexane extractions. For gas- chromatographic analysis these acids ~ere meth.vlated by refluxing I h with I,t, H~SO4 (v/v) in methanol.

Thin-laver chromatography Thin-layer plates (20 c m x 20 em or 20 cm x IO cm) were coated with either

o.25 mm (silica gel-G, Merck) or o.5o mm (Adsorbosil -z, Applied Science l.aborato- ries, Inc.) of adsorbent. The thicker layer was used for preparative purposes, l 'hytol and dihydrophytol, which move together and just ahead of the C~7 sterols on t l , , indicated absorbents, were separated very welt on the AgNOz--silica gel-G system t~ with chloroform-acetic acid (199:1, v/v) (Fig, 2). M:thyl phytanate was resolved frtml some related methyl esters by thin-la3er chromatography on silica gel-G with either light petroleum ethyl ether (I95:5, v jr) or hexane benzene (4o:(ro, v/v} (Fig. 3)- The separation of methyl phy~anate from methyl stearate is impr~ved by applying a small .sample load with maximum length of development.

Sel)arated components on thin-layer plates were located by spraying ~i th either o.5° o, 2,7-dichlorottuorescein in ethanol, in the case of preparative plates and plates f~r autoradiography, or chromic acid and charring ~5. Radiograms were prel:,'t- red with East:nan Kodak Single-c~ated, Blue-.qensitive Xrav Film.

Gas chromatography l ' reliminarv analyses of phytol and its derivatives were made with a dual

c . lumn (4 tt "< 0.25 inch diethylcne glyt'ol succinate) Loenco Model 9oA iastrument

' A

i ! 3 . 5

i l ~ ° a ,'~,a5

, L

m i l l

FiR. 4- Gas chromatograms. _\, unsaponihablus oi runlcn lipids. F.. Jl-tt.tradccanol li}, Jl-hcx,tt|t'- c~xnol (2), dihydrophytol (3}, phytol '~4) anti n-octadecanol (5). C, trimuthylsily| derivatives of cotlll~onent~ in the' unsaponifiables of runletl |ipids. l), trimethylsilyl derivatives of the c~)m- pounds in .%c~m 1~. Apiczon-I. 12olulilll ~'lt 2 4 ~ .

Biochim. 13iophys. Acta, Iz5 {~966l 22-32

20 S. PATTON: A. A. BENSON

equipped with hydrogen flame detectors. This instrument was also fitted xith a high temperature ion chamber-vibrating reed electrometer assembly (total instrumenta- tion by Loenco Inc.) which when used in c(,njunction with thermal c')nductivity detectors would measure both mass and radioactivity in a sample. This system helped us assess radio purity of the synthesized uniformly ~Cdabeled phytol (Fig. ~). In the figure the slight difference in tins)lion ()f tile peaks is due to offset of the two pen:< It: the remainder of the work a Barber Colman Model I() instrument with argon ionization detector was emph)yed.

I t was noted t h a t phy to l anti h)ng-cl,ain ah-oh()ls in general tend t() in t ( , ract wi th polyes ter coat ings . This reduced sens i t i v i t y and made quan t i t a t i ( )n difficult . Thu ; in the l a te r s tages of t he s t u d y an 8 ft /. o.25 inch Apiezon L co lumn wi th -o% coaling on 8o-Ioo mesh Anakr ,ml A B (Analabs , Inc.) was used and p r o v e d ideal in severa l respects . This co lumn when o p e r a t e d a t 25(> a n d 3 o Ib / inch : ar~:(m could be sati~,fact()rily appl ied to the sepa ra t ion of phy to l and relat , d c o m p . u n d s (Fig. 4B), t r i m e t h d s i l v l de r iva tes ()f such c o m p o u n d s (Fig, 4D) and m e t h y l phy ta ) ;Ire (Fig. ()}.

Methy l es ters of f a t t y acids v e r e ana lyzed ma in ly o n . () ft . o.25 inch co lumn con ta in ing a pack ing ,)f 8o-9o A n a k r o m ABS c()ated wi th ~5",, d ie thy lem'g lyco l succ ina te po lyes te r (Analabs . lnc.~, Q u a n t i t a t i o n ()I I)hyt()l a n d (ti l ,y(lr(q)hytol wa,~ accomt) l ished b y l , repar ing ea l ib ra l ion cu rves re la t ing peak art,as lr()m gas chr (m)a tog- r:.tll1:, to (OllCentl;tti()n. I t was norA,d t h a t l)hyt()l gave (rely ()7"- ()f t he re~p,)nse t() the ,h ' tcet( ,r y i d d e d t)v dihy(lr()l)llytol. Smal l g la-s U - t u b e t ra i t s t)a('ked w i lh ~and were used a t time> (m the h e a t e d ()uth' t [r()m gas C]lrt)nl~ttogral)hv e()lmnn> t() ~ ,cover conll)OllelltS f()r r a d i o a c t i v i t y ass;i, ' ()r ()tiler analvs(.'s.

t ~( . l s sav In tile e x p e r i m e n t s wi th unif, rmlv " ( ' - Iabeh ,d phvt,~l, ra ( l i , ) : . ' t iv i tv was nn 'a-

surc( l l )yc( )unt ingv<i th a l)a( 'kard Tr . - ( ' a rb Model 3I 4 F X liquid . ( in l i l la t i ( ,n .i)(.ctr()n~ - eter . ,%;),lllple c()unt> were c()rr( 'c:ed t()r 1)~ckcr()un(1 I)ut ii()t f()r e|:tici(,ll,.'v ({)7')o). Ac t iv i ty in t r aps fr()m tl)t' gas chr()mat()gra )1) ~va~ r(.c()vervd ])v t lushin~ wi th ~cin- tilklti()n llui, I in t , ) c()unting vial-. R a d i ( ) a c t i v I v ()n th in - l ave r l ) late , wa~ a~-av( 'd b o t h by a,~tova0i()gral)hy a n d l)y ~er,q)ing area> (,[ ad-()rl),.,nt in to vial~, tilling th(,se wi th fluid m~d c()unting them.

it;cu.r,,di(m o f vl:iformly labdcd ~'(" pl(vhd ~, ith rmm'n im:c.~ta "r()wztr([ t )ntirlning tlu, t)r(.s(.ncv m . l lll(.l',t])()]i( rclati, )n>hi I) ()/ l)hyt()], d ihv-

dl() l)hytol and i)hytani," acid in the rmnvn , ) q pltyt()l wit- i m u l ) a t v d wi th runlen inges ta and ]abel ing ()f tl,(, (~.nll)(~un(b. iI~ qu,.>li(m wa~ inve>tigatt '( l , l ( ) /H()fan ethan()l ~.()lution ( ( )n ta imng lo4()o() c(mnts rain ()t thv tra( er ~va~ added t() 54 R ~)t freshly ( h a w n rumen in~e~ta in ;t 25o-ml .u.:ti()n fla<k. V a c u u m (It) -() nun l lg) wa.~ d rawn (m the flusk which t hen was ,,eah,d ~-iad i n (u lmted , wi th peri()(li( gent le shaking , fi)r 2 !i a t 37". Purl)ose of the vacuun l w;.s t(~ , ) . ( 'hi t 've al l a l laer() l ) i t" c(m(lit ion, as n() rmal lv plevai]~ in the rklolen, and D) t ' l l t 'ourage a hea(l sj4t('e a t l l losphere ])v f(.rnl(,ntati()rl re.~(.nlblirlg t h a i (ff t he rum(.n. Fc)ll()..ving in(ul)ali( n, lil)ids w(.r(, re('()ver(-d and the 1)resence of an d ac t i v i t y ,)f '~:" in dil vdr()phyt,) l ax(l I )hy tan i ( acid ~va. l)ur~ued I)v the meth()ds 1)revi()u>ly d(,s.ril)ed.

Ittoclam. 13iophys. Acta, I=)5 (l()()b) _)2-32

PHYTOL METABOLISM IN THE BOVINE z7

RESULTS

Phytol and its derivatives in the rumen When the unsaponifiable matter of rumen lipids was analyzed by gas chromat-

ography on the Apiezon column, two prominent peaks of only slightly different re- tention times were observed (Fig. 4A). The faster oI these corresponded to dihydro- phytol, the slower to phytol. When the unsaponifiables were separated on a thin- layer plate (silica gel-G and light petroleum (35-6o°)-ethyl ether-acetic acid (90: I o : I v/v)), and the area corresponding to phytol and dihydrophytol extracted and the extract then gas chromatographed, the same two peaks were observed, l reatment of this extract to form trimethylsilyl derivatives yielded two peaks (Fig. 4C) whidl coincided precisely in retention time wittl those for the dihydrophytoi and phyto| derivatives. It was reasoned that the extract from the silica-gel plate, whic5 does n(,t separate phytol from dihydrophytol, should produce two spots corresponding to phy-

18 17 18

--;--_

• ...... |-- 1

CARBON .NO.

Iris, 5, Thin-laver chromato~cram, .%. fraction extracted frunl tilt- phytol--dihydrophytol area ot , thin-layt.r separation o[ rumen-lipki un~aponiliables. B. phytol and dihydrophytt~l. C. dihydrophy- t4fl. I). phytol. Silica gel A~:Nn~ and chloroform acetic acid (190: ~. v/v).

Fig. f~. (;as chromtt tograms of methyl esters t rapped in the rctcntion zorn, of C-17 on the po].y~-ster column. I 'pper . t rapping from rumen lipid moth.el esters reanalyzed on the polyester column 17-' • Middle. same sample analyzed on Apiezon-L column, z48 . Lower t rapping from blood lipid nlethyl esters (same animal) analyzed on Apiezon-L, z48:. This latter sample from blood lipids yielded da ta from the polyester column closely resembling the upper sca~.

Biochi~n. Biophys. Acta, 125 (t96b) aa-32

2~ ,~. I'AI'[(~N, A. A. BI,,N%(~N

tol and dihydrophy-tol ~ hen chromatographed ~m silica gel containing AgNOa. This was th,' ease (Fig. 5)- Material in the individual spots from the AgNO~-impregnated plate gas c!mmaatographed in proper coincidence with phytol for the ~me spot and dihydr~qflLvtol tor the other. In smnna rv the data were entirely consistent with the presence of both phytol and dihydr(;phyt~l in the rumen contents. Analysis ot a secured sample tr~m another cou welded the same tindings..qtearyl alcoh~fl which tends to chromatograph with phytol or its trinwthyisilvl derivalive (Fig. 4) was sh~wn to be absent by gas chromatographic analysis of tht ~ phy'tol-dihydrophytoI fraction ~m the polyester column. Although this column gave t ~ r lwak resoluti~m for long- chain alcohols, its more t~lar character shifted the retenti~m of stearyl ah'oh.l t~ a positi~n between the phytol and dihyropl,ytol peaks. [ 'nder tht'~e c-ndititm~ the rtl- men samples sh~wed n~ peak e(,rresp~mding t~ st(,arvl alc~hol.

The presence ~f i)hytanic acid in the tattv acids iroln the sap~mified runwn lipids was explored. Methyl esters of the acids fmn~ x~.o mg o~ rtmwn lipid, wt.rt. prepared. Authentic lnethyl phytanate exhibited carb~m llunll~ers ~f lt~.l~ a n d I7. 5

| tx B C

Fig. 7. Au to rad ]ogram from a th in - l ayer : epa ra t i~n of r u m c n lipid unsaponit ial ,h, mat ter , l:n~nt (IL ~,ihydr,~phy'tol (z). phy to l (3), and or igin (4). Silica gel .stgN(~ a n d chh,r~,fornl acet ic acid (t~m: ~ v/v). Fig. 8. Thin- laver ch r fmla togram (light backg rmmd) and aut~radic~gra I~ th'rixed fronl ~t (dark Ilackgt~llnd). :\, rllethvl esters of f a t t y acids with a ca rhon lltllllbt,r o[ 1 7 [rlrln rllnwII in[~t'sta Itl- cuba led ~ i th ~l~f,'iphytot (see als~ upper and n~iddh'scans, Fig. ~0. ]L Ill~'tll~'l phyt~Hlate. C, ntetll~'l stt, a ra te . Silica gel (; a n d light pe t r , ) l eum e thyl t, ther (1~15:5, v/v),

14tuchitjt. Biophys. A~ta, t25 (19tJ6) 22-32

l ' l t V ' l ( ) l . M F . T A I i O L I 5 M I N T H E B O V I N E 29

on tile polyester an~l Apiezon-I. columns respectively, which is in keeping with pu- blished values',~,L The.~e values arc remarkable for a C2o acid and are highly useful for identification purt~)~e~. The methyl :sters from the rumen lipids were then separa- ted by gas chromatography ~m the polyester column and the material elating in the C-I 7 area was collected by a serk-.; of trappings. Gas chromatograms (Fig. 6) of the collected sample on the l~)lyester and Apiez~,n c~)lumns clearly show that a major por- tion of the sample exhibiting a carbon number , f 17 on the polyester colunm eluted at a carbon number of x7.5 on the Apiezon column. In addition to these characteristic carbon numbers, the trapped methyl ester fraction also revealed a component with tt~e same R~. on thin-layer chromatography as that of methyl phytanate (Fig. 8). Tile fatty acids of the bovine blood sample also contained phytanie acid at about the .~ame trace level as found in the rumen lipids {Fig. 6 and Table I).

T A t',l,l-[ I

(i-Vl-.i.% 1)1: I ,HYII ) I . , l l I I I V I . R I I I ' I I Y I I ) I . ~ g l ) I ' I I Y T ~ N I ( A( I() IN I'kV() ~.%51111.1% OF l~('~.li£N INIi I 'SI "~ ,~.NI) ( )N I ' : ~ ~.MI'LI. I)~" | i l l ) I l l )

( . 'oa I (_',q( 2

I.I | )I(I [t~ l()t) l l l l ) ()-()5 (-)-7 (I

I ' h v l , ) l ( " , , , , f h i ) ida , l, [ r t .u () . l(~ ,).o¢, I )l)llll(I ( )...~0 O. l j

l ) q > l ) ~ ' , h - ( , p h s l {",, ~)l h l~id~}, ~rcv (,.2,) ~>.I,) i ) l ) t l n l | Ill[ n i l

I ) h y l l t l l l t i t t t l I * I ' t t l l l t ' l l ( ) .Oj t).()2 | ) h ) o d O o I

o " , , (it ~ l l l t i l l i i l t t V i|(_'ld:'i l l l( ' i i rqlrt~ll i | t , I l l t ' t l l v ] t ' ~ t t ' r ~ |LV ~il~i t hr( l l l | ;_ | loj{Fil i )h.v,

Further evidence 'chat phytol, dihydrophytol and phytanic acid are present in rumen ingesta was secured in the tracer experiment discussed subsequently.

Using the peak-area quantitation described under methods, we determined levels ()I phytol, dihydrophytol and phytanxc acid in tile lipids extracted trom the samples of rumen ingesta. These data (Table I) show that all three compounds occur in very h)w concentrations (,~ I')~, of the lipids). Analysis of tee phytol- dihydrophytol fraction isolated from untreated rumen lipids by thin-layer chromatography enabled estimation of free and bound form.~ of tile two compounds. Results for the two samples show that essentially all of the dihydrophytol anti about one-fi)urth of the phytol are f r e e .

Mel,tbolism of ~(']phytol in the tureen In order to confirm further tile indentificati,)n of dihydrophytol and phytanic

acid and the implication that they are formed f i lm phytol in tilt, rumen, the pre- viously described tracer experiment with :'~('~phytol was conducted. A portion of the unsaponifiables from the total iipids was separa ted by thin layer chronmtography on Adsorbosil-z with the light petroleum- ethyl ether-acetic acid solvent. The phytol- dihydrc)pnytol traction was extracted from the appropriate area of the plate. Alter eonct,ntrating the extract, a portion of it and appropriate reference compounds were

Bioihl)iz. 13iophys. .qcta, 12 5 ( t()o()) 2 z 3 2

3 ° S. PATTON, A. A. B E N S O N

chromatographod on the AgN%-thin-layer system. An autoradiogram (Fig. 7) from this separation clearly showed spots coincident with phytol and dihydrophytol.

Of the radioactivity associated with methyl esters ot fatty acids from the rumen lipids, about oJ 27 Jo could be recovered by trapping a component emerging trom the gas chromatograph at the carbon number for methyl phytanate on the polyester column. This component chromatographed with authentic methyl phytanate on the appropriate thin-layer system from which an autoradiograrn was derived (Fig. 8). Cochromatography of the two components was also demonstrated on the autoradio- gram but the spot was too faint for reproduction.

Incidental to these analyses, quantitation of activity in the various fraction¢ was made (Table ti).

T.~.BI_E 11 RECOV~;R't" OF 14C ACTITVITN" FOLLOkVING INCUBATION OF RUSIEN INGEST.t~ V¢ITH UNIFORMLY 14C'LABELED P H Y I O L

.4 el i;'it V

C~tolts]min %

[ ;nsaponi£abl~is: - : - : - :v.y.,/ ~o ~oo 4.~ dihydrophytol 15800 ' 4 u n i d e n t i f i e d 32 5 0 0 2 S

Sub total 98600 S 5

Fatty acids: phytanic acid 480o .! unidentified t 2 300 , t

~ub total x7 ion 15

Total recovered ~ t5 700 I t t Ac tivitv ad:led i 35 ooo I oo

DISCUSSION

Ruminants normally consun'e phytol in either free or combined form (chloro- phyll). Our findings indicate that free phytol in the rumen is hydrogenated to form dihydrophyt d and is hydrogenated and oxidized to t'~rm phytanic acid. A number ot routes from phytol to phytanie acid are possible. Hydrogenation could occur before or after th~ oxidations at C-I , alcohol to aldehyde and from aldehyde to acid. Our present cursory evidence is that ,~t least part of the phytanie acid occurs in the flee fat ty acid fraction of the rurnen lipids. Further study of the nature and ~;ies of the p~,ssible mechanisms will be required, cspecialfy the extent of microbiologicM involve- l r l~Hl t .

Since the early work of I{I¢ISER TM, it ha~; been firnfly estabfish~ d that the rather highly unsaturated dietary lipids, par teular l 3 linoleate and linolenate, ~r ;.drogena- ted by rumen microorganisms to yield mainly stearate. Thus hydrogenatmn of phytol in the rumen is not entirely unexpec:ed; although it may be instructive regarding enzymic mechanisms and specificity. DUNCAN, GARTON AYD LOUGHG,~, ~, have s F,culated that phytol in feed may give rise to phytanic acid via hydrogenation. However, the 3 seem to suggest that oxidation of dihydrophytol to the acid occurs beyond the rumen. This is reasonable in light of the very low Fh in the rumen, but is not ,xelusively so in light of our findings. The level of phytanic acid we found in the

Btochim. Biophys. Acta. xz 5 (i966) 22-32

PHY'IOL METABOLISM IN THE BOVINE 3"

blood lipids (o.ox%) was of the same order of magnitude as that in the rumen lipids, o.o2-o.o3% and as the 0.05% reported in milk lipids 4. Our efforts to detect phytol or dihydrophytol in cows blood (2 s~mples) or milk fat (6 samples) were unsuccessful. Yet an explanation reconciling the disparity between the levels of phytanic acid in cow's plasma (DUNCAN ANY GARTOS' found 43% in phospholipids) and those reported herein is difficult at present. Three explanations occur to us: (r) large individual animal variations in rumen phytanic acid formation involving teed, rumen microflora, etc. ; (2) a second mechanism of phytanic acid synthesis beyond the lumen; (3) a metabolic block beyond the rumen in some animals which promotes accumulation of the low levels entering the olood from the ingesta.

Some insight to thi~ matter may be provided by data on optical activity re- ported for various sources of phytanic acid. Phytot has three asymmetric carbons (C-7, C-xx, and C-x5) and hydrogenation produces a fourth (C-3). Phytanic acid has the same four asymmetric carbons and variations in its synthesis (and hydrogenation) could affect optical activity. HA,~SEN 18 reports that the optical rotation of Fhytanic acid from butter fat is very similar to that of the same acid from tissue of humans afflicted with Refsum's syndrome. On the other hand, LOUGH indicates his phytanate from cow plasma lipids was at variance in optical activity from that of the acid in butter fat. A proposed alternative route to phytanic acid from farnesoic acid in the liver' may explain the source of LOUGH'S plasma phytanate. However, HANSEN'S observation suggests that the Refsum patient and butterfat phytanate are either preduced by the same mechanism or originate from the same site. The current evi- d, mce ~ suggests that both phytol and phytanate in the diet could be sources of the a~:cumulated phytanate in patients with Refsum's syndrome.

Such factors as the substantial levels of phytanate which can ~ccur in the bo- y ne', the scarcity of information on leveh of phytanic acid in foods, the unique physical properties of the acid including steric effects of the C-3 methyl group on ft..oxidation or lipolysis, and the implications of phytot and phytanic ac'M to human h..'alth define a need for further research.

ACKNOWLEDGEMENTS

We thank E. M. KESLER and staff for obtaining rumen and blood samples. We are indebted to Dr. H. HAVERKAm' BEGE~tAXS for a sample of methyl phytanate and t(, Dr. R. LEwis for technical assistance in instrumentation. The investigation was mlpported in part by U. S. Public Health Service grant HE 03632 and National S,:ience Foundation grant GB 59o.

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