- Email: [email protected]
Studies on the distribution of free carnitine and the occurrence and nature of bound carnitine
ARCHIVES
OF
Studies
BIOCHE!!XISTRY
on the
AND
Distribution and
MYROT\’ From
BIOPHYSICS
98, 146-153 (1962)
of Free Carnitine
Nature
of Bound
iz. MEHLMAN”
AND
and
Carnitine’ GEORGE
WOLF”
the Radiocarbon Laboratory and Division of Animal University of Illinois, Urbana, Illinois Received
March
the Occurrence
Nut&ou,
9, 1962
The distribution of carnitine in various tissues of the rat was investigated, using modifications of existing assays. Muscle showed about 120-180 pg./g. wet weight; brain, 20 pg.; liver 60-90 pg.: testes, 115 pg. Various treatments with organic solvents and acids released no increased amounts of carnitine. All carnitine was dialyzable. No “bound carnitine,” therefore, was detectable in mammalian tissue. This result was confirmed by injection of labeled carnitine, using a radiochemical assay, in combination with the chemical assay. Developing chick embryos showed a great increase in rarnitine between the 12th and 16th day (about 175 and 850 pg. per whole egg, respectively). It was found that yolk sac contained about 4&50% of the carnitine of whole egg, and that some of this, about 2045%, was in a bound form, released by acid treatment and nondialyzable. Specific extraction procedures, followed by hydrolysis, showed this “bound carnitine” to be combined in phospholipid. This finding was confirmed with labeled carnitine. Labeled phospholipid was extractable from the yolk sac, and was purified by silicic acid chromatography. .4 labeled carnitine-containing phospholipid emergrd with the lecithin fraction. The hypothesis is proposed that this material is ohosnhatidylcarnitine. INTRODUCTION
The study of the function and biosynthesis of carnitinc, begun previously in this laboratory (1)) can be cont,inued effectively only if exact information is available on the distribution of t,his substance in the free state, and bound to tissue constituents. Claims have been made for t’he occurrence of ‘(bound carnitinc” by Strack et al. (2) who observed 1040$, of muscle or liver carnitine to be released after HCl treatmcnt (no experimental data are given). Hosein (3) found evidcncc of rarnitine in t,he form of a coenzyme A ester in brain. On thcl other hand, J,indstedt and IJindstcdt 141 ’ Supported by U.S. Public Hralth Grant 8-493. Prescxnted in part at the 46th Annual Meeting, Fetlel~ation of American Soci&es for Experimental Biology, Stlantic City, 1962. (’ Prc,smt address : Department of Nutrition, Foot1 Science and Technology, Massachusetts Institutp of Tec.hnology, Cambridge, Mas.s.
observed that, injected labeled carnitine is not transported to the brain. Adams et al. (5) showed that, carnitinc does not occur bound in serum phospholipid. In the present paper, a quantitative survev is presented of the amounts of free carn&c in various tissues, as ~11 as the results of a search for “bound carnitine.” Fraenkel (6) has demonstrated the dramatic increase in carnitine within a matter of days during development of the chick embryo. Thercforc, the developing chick embryo appeared t.o be an ideal system for a study of carnit,ine biosynthesis. No extraneous (dietary) source of carnitine could interfere with injectw~ precursors. Hence a st,udy of the carnitine content of the chick embryo and yolk sac at various stages of development is here presented, as well as evidence t,o show that yolk sac is the only tissue of those studied in which camitine is found to be bound in phospholipid.
146
\\‘hilc this \I;ork was in l~rog~‘cs~, Lintlstedt and Lindstedt~ (7) dcn~onstrated that y-hutyrobetaine is :t prccurxor of carnitinc in the rat. ESl’ERIMEKTAL ANIMALS;,
DIET
.4m
X~TERMW
Rat :, WPW rnalr albinos of thr Sprague-Dawley struin, kept on :i l:~bolat.ory rhcckers diet. Thaw wwivmg carnitine-C” had been kept, on a carnit in+fwr dirt (1) from wxning. Eggs, which wrw from c.hirk+,ns of the Lrghorn strain, were at 37”. uI.-Carnibine 1~ydrochlo~ideincubntetl ( 146.9 pc../mmole) was prepared as mdhyl-C” tlrwribrtl (1). Lnbrled choline, brtaine, glyvinc. anti algal l)rolrin hydrolyzat c wcw l~nrc~hnwti from the 9urlrar Chicago Corporation. ASSAY
METHODS
Thp assay uwd was a fnrther modification of that originally de~rloprtl by E’Aedman (8) and subsecluently modified in the authors’ l~tboratory (1). The following changes wew made. The we of acrtyl rhloritlc in ethanol :u a vat alyst in the rntrrifiration of v:unitinc iniprovrtl thr rrprocluriI)ility of thr assay. IA?r3 was tliatillctl brfow rlae, EDC WIS distilled from solicl IiOH and zinc. tluat twfow ,w. .4 romplrtr dfwl~ipt ion of thv :ws:q follows.
Purification
on IR-120
Thr tissue to tw andyzrd ww homogenized in thwr timw the vohlmr of 10% TCA, nllowed to stand in thr TCA, filieretl, and wxshrtl, and thr filtratr was rstrnrtrtl with etlret, to remove the, TCA. A enmplc of the filtrntr (pH 2-4) (40-100 ml.), containing betwcrn 20 nntl 500 Fg. carnitinc. wa;: lAxed at thr 101) of a rohlmn (1.1 X 35 cm.) of lR-120 (H’) ion-rschangr rw111, whirh mis then w:rshrd with 200 ml. wit rr, :mcl the sample was dutd with 200 ml. of 1.5 .V ammonia. Thp rluntr ww cY:l~wr:itfd to dl~ynees untlrr rrdllrrtl prc‘.%luv. :incl thr I~cduc~ wm t:lkcLn 111’ in 10 1111. watrr.
Purijicu tion on IRC-50 -4 cohmn (0.8 X 17 cm.) of IRC-50
(H’) ionexrhnngr resin wits eqnilibratcd with 1 J[ phop])htt~ b~ff~~, pH 7.3 for 2-6 In., and wvidred Tvith 10 rd. IKI~PY. The elwtte from t,he IK-120 column (:lbo\-r) \Ws thrn plared on thr c.ollmlrl nncl ’ Abbrrviations E:DC”. rthylcnr ;lCitf.
~wrd: I.A.4, l~oamyl :ilrohol tlichlot~itlr : TC.1. trirhlortrnrrtir
:
148
MEHLMAX
AiYD
WOLF
the IR(1-50 eluate n-as furt’her purifictl on IRA-400. ITsing labeled carnitine, togcthet with Iabelccl betaine, choline, and anlino acids, a single radioactive sl)ot, correspontling to carnitine, was fountl after papcl chroniatograpliy of the IRA-400 eluate. .i c.oltttttn was pwparcvl of 10 g. silicic acid, sudTherefore, the cheuiical assay was routincl> ~wttdfxl in redistilled c~hlot~ofornt contxining 2% pcrforined vn the IKC-50 ctuatcl, tlic ratliotttr~thanol. The column ww ihcn ~v:t~hcd with 250 ml. c~ltloroform-methanol (1 : 1 \-/I-). followed b.x chemical assay on the IRA-400 eluate. It 250 tttl. chloroform cant aining 2% nif~tltanol. ~1 should be noted tha,t the colorirnetric assay could not be carried out on the IRA-400 s;ttttplc of the phospholipid extract etl ;ts described 3bO\~P, in 5 ml. chloroform, x:ts placed on the eluatc, because this resin, when in the OIJ-colutttn nnd eluted with chloroform containing forui, releases quaternary bases which give mcrrwing amounts of methanol and witf~r as I blue complex with brorno~plicnol blue. shosvtt in the rlut,ion graph (Fig. 2). Phosphate wxs determined on all fractions by the tttrthod of Fiske rind SubbaRon, (10) (IJig. 2A).
ASSAT The assay method for carnit,ine of Friedman 18) was further modified, in order to achieve better reproducibility. It was possible to make radioact,ivit,y determinations on the same samples as were used for t,he colorinletric assay. Scintillation counting could not be used because of the severe quenching by the blue color of the carnitine esterbron~ophenol blue cou~l~lcx. However, this complex could be counted in a gas-flow counter satisfactorily when plated “infinitcly thin.” With nietliyl-labeled carnitine, it, was found that consistently only bctwcen 63 and 68% of the radioactivity was ext,mctable into EDC-IA-1 as the carnitine ester-b~oi~ioplienol blue conlplex. The reason for t,his was not explorcvl. It seems likely that, this amount, merely represents the extent of the reaction between the carnitine ester and the dye. The radiochemical purification of carnitine was tested with a niixture of unlabeled carnitine and labeled brtainc and choline. It bccanlc evident, th:it the IRC-50 column satisfactorily remoretl the choline and that brtaine is not taken up in the EDC-IAA. With unlabeled carnitinr and a luixture of labeled glycine or labeled amino acids, there were still some radioactive contaminants which entered the EDC-TAA, along with t,he carnitine, though they did not intcrfcre with the color reaction. To ~niovc these.
DISTRIBUTION IN TIME RAT
The distribution of free and bound carnitine in various tissues, obtained bv various cxt,raction methods, is illustrated in Tables I and II. It can be seen that no lnethod of extract,ion gave significant increases in extractable carnit,ine beyond water or lo’/: TCA extraction. All carnitinc was removable by dialysis after homogenization of t,he tissues in water (Table IV). Therefore, no bound carnit,ine was denionstrable by an) of the nict~liods used, in an? of the tissues tested. This finding is particularly significant in regard to brain: Hoscin (3) claims t,h:tt, this tissue contains carnitine bound in the form of a coenzynie A e&r. No evidence for this form of bound carnitine was obtainable; hydrolysis of brain cxtrmt, witlk HC,‘l gave no increase in carnitinc. However, brain tlocs cont)ain considerable amounts of free cnrnitinc; and labeled carnitinc, wticn irijcctcd, is transported to the brain, a fintling which differs front that of I,imlstcdt antI LintlstecIt (4). The high level of carnitinc in testes is notcn-orthy and may 11s~~ funrtional significance. As ran l)c seen from Table II, the carnitinc content, in tissues of rats which obtained radioartivc carnitinc is consistrntlv lower t,han that of untrcatcrl rat:: iexcept ;n brain). In order to prevent untlue dilution of the radioactive ~losc, rats ust~i in t,hc radioactive exporiulents had been raised on a carnitine-free diet II). This finding is in agreenicnt, with that, of Fraenkel ill) , who observed rat, liver or niuscle from animals fed a rarnitine-free diet to have lower values than those fctl :l normal diet.
FREE
AXD
BOUND TrZBLE
~ISTRIR~TIOS
Carnitine T\vo or more figures shown represent
OF
repent
IN
1)~ various analyses
THE
R.il
procedures
on separate
tissues.
Brain
~luscle
gg.,g.v’el b’l.
p,y.3’s.w”f i(1. 120, 121
PKKdUlY
TCA (Z hr.),’ 'l-C.4 (6-12 hr.),L TCil (i2 hr.)r1 55,;. Chloroform + HCI (1216 hr.)” H2O + HCI( H& onI3 TCA + HCl (2 hr.)” TCA + HCl (18 hr.)d TCA + HCl (72 hr.)” TCA + HCl (0.2 N and heated 18 hr.)d. e
I
CIRNITIKE
isolated
II!)
CARTITI?;E
20, 20 21.0 16, 21.1
r.iver pg.3g u,etwt. x4 90, 91
126 132. i-1
19.5, 21.0 18.T 18.0 20.4 -
135, 130. 132 207, 150, 181, 193 120, li0 -.-
57, 50 61, 65 115 80, 77
148
I* Homogenized t’issue allowed to stand at 4” for times shown in 109, TCA, 5 ml./g. tissue. h Extraction of tissue according to Hosein (3), followed by treat~ment of extract with 0.1 ,V HCl for time shown. c Homogenization in water, treatment of aqueous extract with 0.1 .V HCI at 100” for 30 min. d Homogenization in 10% TCA, followed by treatment of extract with 0.5 .V HCl for times shown. e -4s for footnote (1, hut treatment’ of extract 18 hr. at, 64”. TABLE DISTRIBUTION
OF
II
CARNITINE-C~~
IS
THE
RAT
Pvlethgl-labeled carnitine-Cl*, 21.4 ~c., was illjetted in three portions int,o a 175-g. rat (maint,ained on a carnitine-free diet) over a period of 60 hr. The animal was killed 12 hr. aft>er the last inject,ion. First extract)ion: tissue homogenized and suspended 2 hr. in lO(‘, TCA, filtered, carnitine determined on filtrate. Second extraction: residue suspended in lOgi; TCA 24 hr., filtered, carnit,ine determined on filtrate.
Carnitineobtainedin 1st TCA extraction
Tisue
ps,,R, we, wl,
Muscle Liver Brain Testes
82.5 53.0 21.0 155.0
DWRIBUTI~N
:
Carnitineobtainedin 2ndTCA extraction
colrnls/,,iin.l g. wet wt.
36,600 27,100 3,540 32,000
pg,/g,
I
.&ef w,,
2.2 2.7 4.0 11.5
co~,nlslmin.l ,g. led zut.
1,wO 400 0 520
IK DEVELOPISC EGG
Brcause of the slow t,urnovtr of carnitine in the rat (I), a tissue was sought for biosynthesis studies which would rapidly synthesize a relatively large amount of carnitine. Starting from the observations of Fraenkel (6), that there was an approxi-
niately tenfold increase in carnitine from the egg to the fully developed chick embryo, an investigation was made of the increase in carnitine with t,ime of incubation of whole egg, chick embryo, and yolk sac. As illustrated m Fig. 1, the increase occurs dramatically between the 12th and 16th day. Total carnitine per 16-day egg was about 800-900 pg. Fraenkel (6) reports about’ 420 pg. ( l&day embryo) , based on a bioassay. In the course of t,hc extraction of carnitint from developing eggs, it became al)parent (Fig. 1) that. whole eggs cont.ained some form of carnitine released by TCA in 72 hr. but not in 2 hr. Since wl~olc eggs had this “bound carnitinc,” and embryo alone was found to cont’ain very little of it, t,he yolk sacs were investigated. A comparison of the t,wo showed that, about 20% of carnitinc is liberated by prolonged (72-hr.) treatment with TCA above that. obtainable by direct, extraction from t,he yolk sac. This result was confirmed by injection of labeled carnitinc int,o developing chick embryos (Table III). It is of intcrcst to note that injected carnitine becomes “bound,” particularly in yolk ;;ac.
150
2
4
6 AGE IN DAYS FIG. 1. Cnrnit ine (nontent of de\-eloping chick embryo x-x, represents 2-hr. extraction with TCA; O-0, TCA TABLE INuIR~~oRATI~N
12
14
as a function reprwrnts
16 of time of incubation. 72-111,.rxtraction with
III
0~ LABELED CARNITIKE
ISTO CHICK EMBRYO
Ten-dav-old chick embryos were inject,ed on the 10th and 11th days with a total of 833,370 counts/ min. carnitine hydrochloride-Cl” (spec. activity: 374,000 counts/min./pg.) Embryo and yolk sac were was dehomogenized on the 15th day in 10(,& TCA, allowed to stand for 3 hr., and filtered. Carnitine termined on the filtrate. The residue was extrarted with 10% TCA for 66 hr., filtered, and carnitine determined on the filtrate. \Vet weight of material
Total carnitine
Carnitineig. wet weight
Total radioactivity in carnitine
Radioactivity in carnitine/g.
Specific activity of carnitine
cuunts,‘1min.
comtsjntia.,n1g
g.
PP.
lx.
comls/wtin.
1st TC,4 extract Embryo Yolk sac
I-4 39
390 535
27.9 13.7
161,721 215,700
11,551 5,512
111,000 102,300
2nd TCA extract Embryo Yolk sac
11 30
59 118
11 ,310 32,000
810 820
192,850 270,600
1.2 3 .03
In order to explore further the nature of t,liis binding, homogenates of tissues in water were dialyzed, and the amount of carnitinc released upon 72-hr. treatment with TCA in the material retained in the dialysis bag was investigated. Table IV shows that all carnitinc dialyzed out from aqueous homogmates of musclt~, liwr, and brain, but that
“bound carnitine” was retained in the dialysis bag with honlogcnates of yolk sac. These results suggested that the binding is either t,o protein or to lipid. To investigate the second alternative, phospholipids mere extracted from yolk sacs by a method specific for phospholipid (9), using chloroformmc~thanol. It, was found that, upon IICl hy-
FREE AND BOUKD
drolysis of the ext,racted pl~ospl~olil~id, the water-soluble product contained an amount of carnitine (7.7 pg./g.) comparable to t,he “bound carnitine” released by 72-hr. TCA treat,ment (3.5 pg./g.). That the amount of carnitine released from phospholipid is actually higher might be expected: since the TCA is not as effect,ive as HCl in hydrolyzing the phospholipid carnitine. Labeled carnitinc, when injected into developing (‘ggs (Table V) , resulted in labeled pl~ospl~olipid which, upon hydrolysis, yielded labcletl carnitine, assayed by the specific colorimctric reaction described, and identified b? paper clironiatogra~~liy ; the carnitinp being located by t,lic iodine color waction ( 1) , as ~~11 ad by a radioactivity scanner. As a control, 1:~beled carnitinc was atldecl to yolk sacs clftcr incubat,ion of the eggs, but prior t,o homogenization and extraction. In this case (Table V), no radioactivity XIJ cxtractable into the phospholipitl fraction. Further evidence to insure that the cnrnitint K:LS truly bound to phospholipid, and TABI,E I\ DIALYSIS
ES~~ERIMENT
Tissue honlogenized in wat,er, dialyzed 15 hr. Cont,ents of di:ll,vsis bags extracted with 10”; TC4 for tissues shown. wet
Tissue
weight
Time dialyzed
Tp;.in ~
Cnrnitine ‘g. wet wt.
R-
hr.
hr.
a.
hIusclc
7.5 6.5 5. 1 3.5
0 0 15 45
5 5 6 6
67.0 66.0 6.0 0
J,iver Liver I,iver 1,iver
6.5 6.5 3 6 2.8
0 0 45 45
5 5 6 6
55.0 77.0 3.1 0
Brain Brain Brain
1.8 1 .9 1.8
0 45 45
5 6 6
42.0 0 3 .5
32 32 35 32
0 0 45 45
54 54 72 72
7.5 1.1 5 .?I 3.8
15
0
Muscle MllScle MUSCIP
Yolk Yolk Yolk Yolk
SW sac sax sac
Carnitineo
(i50 pi.) :
If St:Lndnrtl
solution
of cnrnitine
tli:dyzrtl.
0
1.51
CARNITINE
IWORPORATIOS
OF
LABELEU
CARNIT~XE
INTo
I’Hos~~HoLI~~ID
Ten-day-old embryos were injected on the 10th 11th days with R total of 594,900 counts/min. carnitine (spcc. xctivity: 474,000 counts/min.j pg.). The cmhrgos were killed on thr 15th day, and phospholipid was extracted as described. and
ClCarnitine from phospholipid WZLSpaper chromatographed (1) with and wit,hout carrier, and radioactIve peak was sho\vn to correspond to cnrnitine. b Carnitine-Cl” (100,000 countri/min.) was added to :I yolk SW clfter incubation \)ut, before homogenization and extraction.
not’ a contaminant, was obtained t)y paper chromatography. The extracted labeled pliosl~l~olipid from yolk sac showcd an K, of 0.56, with the radlonctiw spot coinciding with the phospholipid spot. Free carnitine, in this system (diisobutyl ketone-acetic acid-water, 40 : 20 : 3 ; silicic a&l-impregnatctl Whatman no. 1 papw) rcm:Aned at tlw origin. It can be seen t,llat, the specific activity of phospholipid carnitinc from yolk sac !Table 1-j is close to that of the bound carnitinc released from yolk XRCby prolonged TC‘A treatment (Table III). So phospholipid-bound carnitinc ~15 dctectablc in rat liver or brain. kktrncted labeled pliospl~olipid, obtained from :i tlevcloping egg injected with 1:~bclct-i cnrnitinc (total of 594,000 counts,‘min. ) , was wbjectetl to chromatography on a silicit acid column. Radio:wtiCt~y and phosphate content, of the fractions in the eluate TWL’Cl)lotted as shown (Fig. 2,1). TIE peak for radioactivity followed closclv upon, but was different from, tlic peak for cholinecontaining pliospliolipid~ prrsumnbly lccithin. IIowerrr. as cnn be seen from t,lie graph: the lecithin peak ha(l a considerable o\-wlnp with the radioncti~e peak.
1.52
MEHLMAN
AND WOLF
t 2800 12600 --2400 -~2200 --zoo0 --I800 4600 1400 I50
I60
170
180
I90 Tube
em
210
220
230
240 1200
number
~ 4 : z u
,000 1 800 --600 --400 --zoo
FIG. 2A. Elution of yolk sac phospholipid from a silicic acid column, showing phosphate content (O--O), or radioactivity (X-X) as a function of volume eluted. Tubes 1-18, eluted with 2% methanol in chloroform; 19-35, 20% methanol in chloroform ; 35-50, 20% met,hanol, 1.25% water in chloroform; 51-64, 20% methanol, 2.5% water in chloroform; 6581, 40% methanol, 6% water in chloroform. Inset (Fig. 2R) : rcchromatography of the mdioa&e peak of Fig. 2A.
tive peak, two radioactive peaks were obtained (Fig. 2B), designated A and B. After combination of the fractions of each peak and hydrolysis, carnit,ine was assayed : peak A (12.0 mg. ; 3650 counts/min. j showed no carnitine calorimetrically though the radioactivity was extractable into EDC-IAA. By far the larger peak was B (198 mg.; 26,480 counts/min.) This contained 380 pg. carnitine, which accounted for 47.6% of the radioactivity of the fraction. However, the analysis figures for this fraction corresponded closely to those of lecithin. zlnal. Calcd. for C44HR80RPX: C, 66.88; I-l, 11.21; N, 1.78. Found: C, 66.98; H, 9.90; N, 1.95. We propose the hypothesis, therefore, that this fraction is lecithin or a phospholipid closely similar to lecithin, containing a small amount of labeled phosphat,idylcarnitine. The carnitine cont,ent’ of this fraction was only 0.19%
(if the whole
fraction
were
phosphatidvlcarnitine, it would contain 23.3% rarmtine). In fact, the amount of
carnitine obtained from this fract,ion (380 pg.) is comparable to that generally obtained as “bound carnit,ine” (118 pg.) from yolk sac (cf. Table III). A research project has been underta,ken toward identification and complete structure determination of t,he phospholipid-bound carnitine. It is possible that the material in peak A, above, is the phosphatidylmet,hylcholine, recently reported by Newburgh et al. (12), which they post,ulate t,o be derived from carnitine by decarboxylation. In view of the probable occurrence of phosphatidylcarnitine presently described, it would seem more probable that bhe latt,er is the precursor of phosphatidylmethylcholine, rat’her than free carnitinc. That carnitine bound in phospholipid has bccln detectable only in developing chick embryo points to a twofold function for carnit,inc: one involving the fret compound, as present in muscle, liver, brain or testes; another the phospholipid-hound compound, as found in yolk sac and to some ext,ent in
FREE AND BOUND
chick embryo. Ito and Fraenkel (131 showed, by use of a competitive inhibitor, that carnitine is required for the early development of chick embryo. This may be the phospholipid-bound form. On the other hand, the larvae of Tenebrio! which require carnitine for survival, deem to utilize the free form, since Fraenkel (6) could detcct practically no carnit,ine in yolk sacs of l&day-old embryos, IThen using tlir> growth of t’he larvae as’ a bioassay. Recent evidence seems to indicate t,liat choline can be synthesizetl from cthnnolamine aft’cr the latter has been bound to pliospliolipid ( I4 j . One might similarly suppose that phospholipid-bound carnitine occurs in this form as a result of its bioaynt,hcsis. That this is not so is shown by the fact that injected labeled cnrnitine readily becomes attached to phospholipid. It is therefore likely that carnitinc in this form has some functional significance.
1.x
CARXITIXE
1.
~VOLF, G., .mD BERCXR, C.
R. A., drch. Lliockcuc. Biophys. 92, 360 (1961). 2. sTR.WK, E., ltOTZSCE1, \v., .hKD LOHENZ, I., /hr “Prot KICS of the Biologica f Fluids,” (H. l’ertcrs, rd.). p. 235. Elm% PuMshing Co., Arnsittwfm~, 1960. 3. Hosm, E., :Ytrtrlw 187, 321 (1960). 4. J~ISUS~TEDT, S., ASD LINMTEDT, C;., Acta Cltw~t~. Scntrti. 15, 701 (1961).
9. IhICKSOS,
s.
r:., ASI) 12.4.\DX, E. hf.,
P,'lK.
,%C.
F:xptl. I~ioZ. Ned. 102, 512 (1959). 10. FISKE, c. H., ASD %mhw, y.; J. Biol. t?hm. 66,375 ( 1925). 11. FHIEI\KEL, G., ildc. Biochcm. Biophys. 50, 486 (1954). 12. SEW.UIIH~:R, R. JT., ;\(;.4Rw.-AL, H., BIEBEH, I,.. AXU C~LDELIX. V. H., Fcc(c,rc~ion Pmt. 21, 293 ( 1962) 13. ITO. T., .\.\u I~H.WSE(EL, ci., cf. Gen. Ph~.siol. 41, 279 ( 19X). 14. (;IBSOA,
I<. L).. ~~ILsox,
J. D., AND CDESFRIEND,
S.. J. lliot. C’h~/n. 236, 6i3 (1961).
OF
Studies
BIOCHE!!XISTRY
on the
AND
Distribution and
MYROT\’ From
BIOPHYSICS
98, 146-153 (1962)
of Free Carnitine
Nature
of Bound
iz. MEHLMAN”
AND
and
Carnitine’ GEORGE
WOLF”
the Radiocarbon Laboratory and Division of Animal University of Illinois, Urbana, Illinois Received
March
the Occurrence
Nut&ou,
9, 1962
The distribution of carnitine in various tissues of the rat was investigated, using modifications of existing assays. Muscle showed about 120-180 pg./g. wet weight; brain, 20 pg.; liver 60-90 pg.: testes, 115 pg. Various treatments with organic solvents and acids released no increased amounts of carnitine. All carnitine was dialyzable. No “bound carnitine,” therefore, was detectable in mammalian tissue. This result was confirmed by injection of labeled carnitine, using a radiochemical assay, in combination with the chemical assay. Developing chick embryos showed a great increase in rarnitine between the 12th and 16th day (about 175 and 850 pg. per whole egg, respectively). It was found that yolk sac contained about 4&50% of the carnitine of whole egg, and that some of this, about 2045%, was in a bound form, released by acid treatment and nondialyzable. Specific extraction procedures, followed by hydrolysis, showed this “bound carnitine” to be combined in phospholipid. This finding was confirmed with labeled carnitine. Labeled phospholipid was extractable from the yolk sac, and was purified by silicic acid chromatography. .4 labeled carnitine-containing phospholipid emergrd with the lecithin fraction. The hypothesis is proposed that this material is ohosnhatidylcarnitine. INTRODUCTION
The study of the function and biosynthesis of carnitinc, begun previously in this laboratory (1)) can be cont,inued effectively only if exact information is available on the distribution of t,his substance in the free state, and bound to tissue constituents. Claims have been made for t’he occurrence of ‘(bound carnitinc” by Strack et al. (2) who observed 1040$, of muscle or liver carnitine to be released after HCl treatmcnt (no experimental data are given). Hosein (3) found evidcncc of rarnitine in t,he form of a coenzyme A ester in brain. On thcl other hand, J,indstedt and IJindstcdt 141 ’ Supported by U.S. Public Hralth Grant 8-493. Prescxnted in part at the 46th Annual Meeting, Fetlel~ation of American Soci&es for Experimental Biology, Stlantic City, 1962. (’ Prc,smt address : Department of Nutrition, Foot1 Science and Technology, Massachusetts Institutp of Tec.hnology, Cambridge, Mas.s.
observed that, injected labeled carnitine is not transported to the brain. Adams et al. (5) showed that, carnitinc does not occur bound in serum phospholipid. In the present paper, a quantitative survev is presented of the amounts of free carn&c in various tissues, as ~11 as the results of a search for “bound carnitine.” Fraenkel (6) has demonstrated the dramatic increase in carnitine within a matter of days during development of the chick embryo. Thercforc, the developing chick embryo appeared t.o be an ideal system for a study of carnit,ine biosynthesis. No extraneous (dietary) source of carnitine could interfere with injectw~ precursors. Hence a st,udy of the carnitine content of the chick embryo and yolk sac at various stages of development is here presented, as well as evidence t,o show that yolk sac is the only tissue of those studied in which camitine is found to be bound in phospholipid.
146
\\‘hilc this \I;ork was in l~rog~‘cs~, Lintlstedt and Lindstedt~ (7) dcn~onstrated that y-hutyrobetaine is :t prccurxor of carnitinc in the rat. ESl’ERIMEKTAL ANIMALS;,
DIET
.4m
X~TERMW
Rat :, WPW rnalr albinos of thr Sprague-Dawley struin, kept on :i l:~bolat.ory rhcckers diet. Thaw wwivmg carnitine-C” had been kept, on a carnit in+fwr dirt (1) from wxning. Eggs, which wrw from c.hirk+,ns of the Lrghorn strain, were at 37”. uI.-Carnibine 1~ydrochlo~ideincubntetl ( 146.9 pc../mmole) was prepared as mdhyl-C” tlrwribrtl (1). Lnbrled choline, brtaine, glyvinc. anti algal l)rolrin hydrolyzat c wcw l~nrc~hnwti from the 9urlrar Chicago Corporation. ASSAY
METHODS
Thp assay uwd was a fnrther modification of that originally de~rloprtl by E’Aedman (8) and subsecluently modified in the authors’ l~tboratory (1). The following changes wew made. The we of acrtyl rhloritlc in ethanol :u a vat alyst in the rntrrifiration of v:unitinc iniprovrtl thr rrprocluriI)ility of thr assay. IA?r3 was tliatillctl brfow rlae, EDC WIS distilled from solicl IiOH and zinc. tluat twfow ,w. .4 romplrtr dfwl~ipt ion of thv :ws:q follows.
Purification
on IR-120
Thr tissue to tw andyzrd ww homogenized in thwr timw the vohlmr of 10% TCA, nllowed to stand in thr TCA, filieretl, and wxshrtl, and thr filtratr was rstrnrtrtl with etlret, to remove the, TCA. A enmplc of the filtrntr (pH 2-4) (40-100 ml.), containing betwcrn 20 nntl 500 Fg. carnitinc. wa;: lAxed at thr 101) of a rohlmn (1.1 X 35 cm.) of lR-120 (H’) ion-rschangr rw111, whirh mis then w:rshrd with 200 ml. wit rr, :mcl the sample was dutd with 200 ml. of 1.5 .V ammonia. Thp rluntr ww cY:l~wr:itfd to dl~ynees untlrr rrdllrrtl prc‘.%luv. :incl thr I~cduc~ wm t:lkcLn 111’ in 10 1111. watrr.
Purijicu tion on IRC-50 -4 cohmn (0.8 X 17 cm.) of IRC-50
(H’) ionexrhnngr resin wits eqnilibratcd with 1 J[ phop])htt~ b~ff~~, pH 7.3 for 2-6 In., and wvidred Tvith 10 rd. IKI~PY. The elwtte from t,he IK-120 column (:lbo\-r) \Ws thrn plared on thr c.ollmlrl nncl ’ Abbrrviations E:DC”. rthylcnr ;lCitf.
~wrd: I.A.4, l~oamyl :ilrohol tlichlot~itlr : TC.1. trirhlortrnrrtir
:
148
MEHLMAX
AiYD
WOLF
the IR(1-50 eluate n-as furt’her purifictl on IRA-400. ITsing labeled carnitine, togcthet with Iabelccl betaine, choline, and anlino acids, a single radioactive sl)ot, correspontling to carnitine, was fountl after papcl chroniatograpliy of the IRA-400 eluate. .i c.oltttttn was pwparcvl of 10 g. silicic acid, sudTherefore, the cheuiical assay was routincl> ~wttdfxl in redistilled c~hlot~ofornt contxining 2% pcrforined vn the IKC-50 ctuatcl, tlic ratliotttr~thanol. The column ww ihcn ~v:t~hcd with 250 ml. c~ltloroform-methanol (1 : 1 \-/I-). followed b.x chemical assay on the IRA-400 eluate. It 250 tttl. chloroform cant aining 2% nif~tltanol. ~1 should be noted tha,t the colorirnetric assay could not be carried out on the IRA-400 s;ttttplc of the phospholipid extract etl ;ts described 3bO\~P, in 5 ml. chloroform, x:ts placed on the eluatc, because this resin, when in the OIJ-colutttn nnd eluted with chloroform containing forui, releases quaternary bases which give mcrrwing amounts of methanol and witf~r as I blue complex with brorno~plicnol blue. shosvtt in the rlut,ion graph (Fig. 2). Phosphate wxs determined on all fractions by the tttrthod of Fiske rind SubbaRon, (10) (IJig. 2A).
ASSAT The assay method for carnit,ine of Friedman 18) was further modified, in order to achieve better reproducibility. It was possible to make radioact,ivit,y determinations on the same samples as were used for t,he colorinletric assay. Scintillation counting could not be used because of the severe quenching by the blue color of the carnitine esterbron~ophenol blue cou~l~lcx. However, this complex could be counted in a gas-flow counter satisfactorily when plated “infinitcly thin.” With nietliyl-labeled carnitine, it, was found that consistently only bctwcen 63 and 68% of the radioactivity was ext,mctable into EDC-IA-1 as the carnitine ester-b~oi~ioplienol blue conlplex. The reason for t,his was not explorcvl. It seems likely that, this amount, merely represents the extent of the reaction between the carnitine ester and the dye. The radiochemical purification of carnitine was tested with a niixture of unlabeled carnitine and labeled brtainc and choline. It bccanlc evident, th:it the IRC-50 column satisfactorily remoretl the choline and that brtaine is not taken up in the EDC-IAA. With unlabeled carnitinr and a luixture of labeled glycine or labeled amino acids, there were still some radioactive contaminants which entered the EDC-TAA, along with t,he carnitine, though they did not intcrfcre with the color reaction. To ~niovc these.
DISTRIBUTION IN TIME RAT
The distribution of free and bound carnitine in various tissues, obtained bv various cxt,raction methods, is illustrated in Tables I and II. It can be seen that no lnethod of extract,ion gave significant increases in extractable carnit,ine beyond water or lo’/: TCA extraction. All carnitinc was removable by dialysis after homogenization of t,he tissues in water (Table IV). Therefore, no bound carnit,ine was denionstrable by an) of the nict~liods used, in an? of the tissues tested. This finding is particularly significant in regard to brain: Hoscin (3) claims t,h:tt, this tissue contains carnitine bound in the form of a coenzynie A e&r. No evidence for this form of bound carnitine was obtainable; hydrolysis of brain cxtrmt, witlk HC,‘l gave no increase in carnitinc. However, brain tlocs cont)ain considerable amounts of free cnrnitinc; and labeled carnitinc, wticn irijcctcd, is transported to the brain, a fintling which differs front that of I,imlstcdt antI LintlstecIt (4). The high level of carnitinc in testes is notcn-orthy and may 11s~~ funrtional significance. As ran l)c seen from Table II, the carnitinc content, in tissues of rats which obtained radioartivc carnitinc is consistrntlv lower t,han that of untrcatcrl rat:: iexcept ;n brain). In order to prevent untlue dilution of the radioactive ~losc, rats ust~i in t,hc radioactive exporiulents had been raised on a carnitine-free diet II). This finding is in agreenicnt, with that, of Fraenkel ill) , who observed rat, liver or niuscle from animals fed a rarnitine-free diet to have lower values than those fctl :l normal diet.
FREE
AXD
BOUND TrZBLE
~ISTRIR~TIOS
Carnitine T\vo or more figures shown represent
OF
repent
IN
1)~ various analyses
THE
R.il
procedures
on separate
tissues.
Brain
~luscle
gg.,g.v’el b’l.
p,y.3’s.w”f i(1. 120, 121
PKKdUlY
TCA (Z hr.),’ 'l-C.4 (6-12 hr.),L TCil (i2 hr.)r1 55,;. Chloroform + HCI (1216 hr.)” H2O + HCI( H& onI3 TCA + HCl (2 hr.)” TCA + HCl (18 hr.)d TCA + HCl (72 hr.)” TCA + HCl (0.2 N and heated 18 hr.)d. e
I
CIRNITIKE
isolated
II!)
CARTITI?;E
20, 20 21.0 16, 21.1
r.iver pg.3g u,etwt. x4 90, 91
126 132. i-1
19.5, 21.0 18.T 18.0 20.4 -
135, 130. 132 207, 150, 181, 193 120, li0 -.-
57, 50 61, 65 115 80, 77
148
I* Homogenized t’issue allowed to stand at 4” for times shown in 109, TCA, 5 ml./g. tissue. h Extraction of tissue according to Hosein (3), followed by treat~ment of extract with 0.1 ,V HCl for time shown. c Homogenization in water, treatment of aqueous extract with 0.1 .V HCI at 100” for 30 min. d Homogenization in 10% TCA, followed by treatment of extract with 0.5 .V HCl for times shown. e -4s for footnote (1, hut treatment’ of extract 18 hr. at, 64”. TABLE DISTRIBUTION
OF
II
CARNITINE-C~~
IS
THE
RAT
Pvlethgl-labeled carnitine-Cl*, 21.4 ~c., was illjetted in three portions int,o a 175-g. rat (maint,ained on a carnitine-free diet) over a period of 60 hr. The animal was killed 12 hr. aft>er the last inject,ion. First extract)ion: tissue homogenized and suspended 2 hr. in lO(‘, TCA, filtered, carnitine determined on filtrate. Second extraction: residue suspended in lOgi; TCA 24 hr., filtered, carnit,ine determined on filtrate.
Carnitineobtainedin 1st TCA extraction
Tisue
ps,,R, we, wl,
Muscle Liver Brain Testes
82.5 53.0 21.0 155.0
DWRIBUTI~N
:
Carnitineobtainedin 2ndTCA extraction
colrnls/,,iin.l g. wet wt.
36,600 27,100 3,540 32,000
pg,/g,
I
.&ef w,,
2.2 2.7 4.0 11.5
co~,nlslmin.l ,g. led zut.
1,wO 400 0 520
IK DEVELOPISC EGG
Brcause of the slow t,urnovtr of carnitine in the rat (I), a tissue was sought for biosynthesis studies which would rapidly synthesize a relatively large amount of carnitine. Starting from the observations of Fraenkel (6), that there was an approxi-
niately tenfold increase in carnitine from the egg to the fully developed chick embryo, an investigation was made of the increase in carnitine with t,ime of incubation of whole egg, chick embryo, and yolk sac. As illustrated m Fig. 1, the increase occurs dramatically between the 12th and 16th day. Total carnitine per 16-day egg was about 800-900 pg. Fraenkel (6) reports about’ 420 pg. ( l&day embryo) , based on a bioassay. In the course of t,hc extraction of carnitint from developing eggs, it became al)parent (Fig. 1) that. whole eggs cont.ained some form of carnitine released by TCA in 72 hr. but not in 2 hr. Since wl~olc eggs had this “bound carnitinc,” and embryo alone was found to cont’ain very little of it, t,he yolk sacs were investigated. A comparison of the t,wo showed that, about 20% of carnitinc is liberated by prolonged (72-hr.) treatment with TCA above that. obtainable by direct, extraction from t,he yolk sac. This result was confirmed by injection of labeled carnitinc int,o developing chick embryos (Table III). It is of intcrcst to note that injected carnitine becomes “bound,” particularly in yolk ;;ac.
150
2
4
6 AGE IN DAYS FIG. 1. Cnrnit ine (nontent of de\-eloping chick embryo x-x, represents 2-hr. extraction with TCA; O-0, TCA TABLE INuIR~~oRATI~N
12
14
as a function reprwrnts
16 of time of incubation. 72-111,.rxtraction with
III
0~ LABELED CARNITIKE
ISTO CHICK EMBRYO
Ten-dav-old chick embryos were inject,ed on the 10th and 11th days with a total of 833,370 counts/ min. carnitine hydrochloride-Cl” (spec. activity: 374,000 counts/min./pg.) Embryo and yolk sac were was dehomogenized on the 15th day in 10(,& TCA, allowed to stand for 3 hr., and filtered. Carnitine termined on the filtrate. The residue was extrarted with 10% TCA for 66 hr., filtered, and carnitine determined on the filtrate. \Vet weight of material
Total carnitine
Carnitineig. wet weight
Total radioactivity in carnitine
Radioactivity in carnitine/g.
Specific activity of carnitine
cuunts,‘1min.
comtsjntia.,n1g
g.
PP.
lx.
comls/wtin.
1st TC,4 extract Embryo Yolk sac
I-4 39
390 535
27.9 13.7
161,721 215,700
11,551 5,512
111,000 102,300
2nd TCA extract Embryo Yolk sac
11 30
59 118
11 ,310 32,000
810 820
192,850 270,600
1.2 3 .03
In order to explore further the nature of t,liis binding, homogenates of tissues in water were dialyzed, and the amount of carnitinc released upon 72-hr. treatment with TCA in the material retained in the dialysis bag was investigated. Table IV shows that all carnitinc dialyzed out from aqueous homogmates of musclt~, liwr, and brain, but that
“bound carnitine” was retained in the dialysis bag with honlogcnates of yolk sac. These results suggested that the binding is either t,o protein or to lipid. To investigate the second alternative, phospholipids mere extracted from yolk sacs by a method specific for phospholipid (9), using chloroformmc~thanol. It, was found that, upon IICl hy-
FREE AND BOUKD
drolysis of the ext,racted pl~ospl~olil~id, the water-soluble product contained an amount of carnitine (7.7 pg./g.) comparable to t,he “bound carnitine” released by 72-hr. TCA treat,ment (3.5 pg./g.). That the amount of carnitine released from phospholipid is actually higher might be expected: since the TCA is not as effect,ive as HCl in hydrolyzing the phospholipid carnitine. Labeled carnitinc, when injected into developing (‘ggs (Table V) , resulted in labeled pl~ospl~olipid which, upon hydrolysis, yielded labcletl carnitine, assayed by the specific colorimctric reaction described, and identified b? paper clironiatogra~~liy ; the carnitinp being located by t,lic iodine color waction ( 1) , as ~~11 ad by a radioactivity scanner. As a control, 1:~beled carnitinc was atldecl to yolk sacs clftcr incubat,ion of the eggs, but prior t,o homogenization and extraction. In this case (Table V), no radioactivity XIJ cxtractable into the phospholipitl fraction. Further evidence to insure that the cnrnitint K:LS truly bound to phospholipid, and TABI,E I\ DIALYSIS
ES~~ERIMENT
Tissue honlogenized in wat,er, dialyzed 15 hr. Cont,ents of di:ll,vsis bags extracted with 10”; TC4 for tissues shown. wet
Tissue
weight
Time dialyzed
Tp;.in ~
Cnrnitine ‘g. wet wt.
R-
hr.
hr.
a.
hIusclc
7.5 6.5 5. 1 3.5
0 0 15 45
5 5 6 6
67.0 66.0 6.0 0
J,iver Liver I,iver 1,iver
6.5 6.5 3 6 2.8
0 0 45 45
5 5 6 6
55.0 77.0 3.1 0
Brain Brain Brain
1.8 1 .9 1.8
0 45 45
5 6 6
42.0 0 3 .5
32 32 35 32
0 0 45 45
54 54 72 72
7.5 1.1 5 .?I 3.8
15
0
Muscle MllScle MUSCIP
Yolk Yolk Yolk Yolk
SW sac sax sac
Carnitineo
(i50 pi.) :
If St:Lndnrtl
solution
of cnrnitine
tli:dyzrtl.
0
1.51
CARNITINE
IWORPORATIOS
OF
LABELEU
CARNIT~XE
INTo
I’Hos~~HoLI~~ID
Ten-day-old embryos were injected on the 10th 11th days with R total of 594,900 counts/min. carnitine (spcc. xctivity: 474,000 counts/min.j pg.). The cmhrgos were killed on thr 15th day, and phospholipid was extracted as described. and
ClCarnitine from phospholipid WZLSpaper chromatographed (1) with and wit,hout carrier, and radioactIve peak was sho\vn to correspond to cnrnitine. b Carnitine-Cl” (100,000 countri/min.) was added to :I yolk SW clfter incubation \)ut, before homogenization and extraction.
not’ a contaminant, was obtained t)y paper chromatography. The extracted labeled pliosl~l~olipid from yolk sac showcd an K, of 0.56, with the radlonctiw spot coinciding with the phospholipid spot. Free carnitine, in this system (diisobutyl ketone-acetic acid-water, 40 : 20 : 3 ; silicic a&l-impregnatctl Whatman no. 1 papw) rcm:Aned at tlw origin. It can be seen t,llat, the specific activity of phospholipid carnitinc from yolk sac !Table 1-j is close to that of the bound carnitinc released from yolk XRCby prolonged TC‘A treatment (Table III). So phospholipid-bound carnitinc ~15 dctectablc in rat liver or brain. kktrncted labeled pliospl~olipid, obtained from :i tlevcloping egg injected with 1:~bclct-i cnrnitinc (total of 594,000 counts,‘min. ) , was wbjectetl to chromatography on a silicit acid column. Radio:wtiCt~y and phosphate content, of the fractions in the eluate TWL’Cl)lotted as shown (Fig. 2,1). TIE peak for radioactivity followed closclv upon, but was different from, tlic peak for cholinecontaining pliospliolipid~ prrsumnbly lccithin. IIowerrr. as cnn be seen from t,lie graph: the lecithin peak ha(l a considerable o\-wlnp with the radioncti~e peak.
1.52
MEHLMAN
AND WOLF
t 2800 12600 --2400 -~2200 --zoo0 --I800 4600 1400 I50
I60
170
180
I90 Tube
em
210
220
230
240 1200
number
~ 4 : z u
,000 1 800 --600 --400 --zoo
FIG. 2A. Elution of yolk sac phospholipid from a silicic acid column, showing phosphate content (O--O), or radioactivity (X-X) as a function of volume eluted. Tubes 1-18, eluted with 2% methanol in chloroform; 19-35, 20% methanol in chloroform ; 35-50, 20% met,hanol, 1.25% water in chloroform; 51-64, 20% methanol, 2.5% water in chloroform; 6581, 40% methanol, 6% water in chloroform. Inset (Fig. 2R) : rcchromatography of the mdioa&e peak of Fig. 2A.
tive peak, two radioactive peaks were obtained (Fig. 2B), designated A and B. After combination of the fractions of each peak and hydrolysis, carnit,ine was assayed : peak A (12.0 mg. ; 3650 counts/min. j showed no carnitine calorimetrically though the radioactivity was extractable into EDC-IAA. By far the larger peak was B (198 mg.; 26,480 counts/min.) This contained 380 pg. carnitine, which accounted for 47.6% of the radioactivity of the fraction. However, the analysis figures for this fraction corresponded closely to those of lecithin. zlnal. Calcd. for C44HR80RPX: C, 66.88; I-l, 11.21; N, 1.78. Found: C, 66.98; H, 9.90; N, 1.95. We propose the hypothesis, therefore, that this fraction is lecithin or a phospholipid closely similar to lecithin, containing a small amount of labeled phosphat,idylcarnitine. The carnitine cont,ent’ of this fraction was only 0.19%
(if the whole
fraction
were
phosphatidvlcarnitine, it would contain 23.3% rarmtine). In fact, the amount of
carnitine obtained from this fract,ion (380 pg.) is comparable to that generally obtained as “bound carnit,ine” (118 pg.) from yolk sac (cf. Table III). A research project has been underta,ken toward identification and complete structure determination of t,he phospholipid-bound carnitine. It is possible that the material in peak A, above, is the phosphatidylmet,hylcholine, recently reported by Newburgh et al. (12), which they post,ulate t,o be derived from carnitine by decarboxylation. In view of the probable occurrence of phosphatidylcarnitine presently described, it would seem more probable that bhe latt,er is the precursor of phosphatidylmethylcholine, rat’her than free carnitinc. That carnitine bound in phospholipid has bccln detectable only in developing chick embryo points to a twofold function for carnit,inc: one involving the fret compound, as present in muscle, liver, brain or testes; another the phospholipid-hound compound, as found in yolk sac and to some ext,ent in
FREE AND BOUND
chick embryo. Ito and Fraenkel (131 showed, by use of a competitive inhibitor, that carnitine is required for the early development of chick embryo. This may be the phospholipid-bound form. On the other hand, the larvae of Tenebrio! which require carnitine for survival, deem to utilize the free form, since Fraenkel (6) could detcct practically no carnit,ine in yolk sacs of l&day-old embryos, IThen using tlir> growth of t’he larvae as’ a bioassay. Recent evidence seems to indicate t,liat choline can be synthesizetl from cthnnolamine aft’cr the latter has been bound to pliospliolipid ( I4 j . One might similarly suppose that phospholipid-bound carnitine occurs in this form as a result of its bioaynt,hcsis. That this is not so is shown by the fact that injected labeled cnrnitine readily becomes attached to phospholipid. It is therefore likely that carnitinc in this form has some functional significance.
1.x
CARXITIXE
1.
~VOLF, G., .mD BERCXR, C.
R. A., drch. Lliockcuc. Biophys. 92, 360 (1961). 2. sTR.WK, E., ltOTZSCE1, \v., .hKD LOHENZ, I., /hr “Prot KICS of the Biologica f Fluids,” (H. l’ertcrs, rd.). p. 235. Elm% PuMshing Co., Arnsittwfm~, 1960. 3. Hosm, E., :Ytrtrlw 187, 321 (1960). 4. J~ISUS~TEDT, S., ASD LINMTEDT, C;., Acta Cltw~t~. Scntrti. 15, 701 (1961).
9. IhICKSOS,
s.
r:., ASI) 12.4.\DX, E. hf.,
P,'lK.
,%C.
F:xptl. I~ioZ. Ned. 102, 512 (1959). 10. FISKE, c. H., ASD %mhw, y.; J. Biol. t?hm. 66,375 ( 1925). 11. FHIEI\KEL, G., ildc. Biochcm. Biophys. 50, 486 (1954). 12. SEW.UIIH~:R, R. JT., ;\(;.4Rw.-AL, H., BIEBEH, I,.. AXU C~LDELIX. V. H., Fcc(c,rc~ion Pmt. 21, 293 ( 1962) 13. ITO. T., .\.\u I~H.WSE(EL, ci., cf. Gen. Ph~.siol. 41, 279 ( 19X). 14. (;IBSOA,
I<. L).. ~~ILsox,
J. D., AND CDESFRIEND,
S.. J. lliot. C’h~/n. 236, 6i3 (1961).