~ J B I Q U I KONE-8FROM Plnsniorlio ml). The lower limit of detection for this spray is about 1mcg/ cm2. All of the iemaiiiirig residue of the purified ubiquinone was subjected to high-resolution mass spectrometry? with a Model 20103C inst,rument of the Consolidated Electrodynamics Corp. (CEC), which was modified to provide an increased magnetic field, an electron-multiplier Wien-filt,er detection system, a sampling system permitting direct sample introduction by means of a heated probe, and 1-mm slits to permit unit resolution at m / e 1000. After int,roduction of the sample, it was necessary to hold the sample at a probe temperature of 125' until traces of volatile impurities were eliminated. The mass spectrum, determined at a probe temperature of 380", showed a weak molecular ion at mle 862 for ubiquinone-10 and the molecular ions of ubiquinone-8 ( w e 726) and -9 (m/e 794) corresponding to traces of t.hese ubiquinones in Rhesus monkey blood. A strong molecular ion for itbiquinone-9 at m;le 794, a weak molecular ion at m/e 726 for ubiquinone -8 and a molecular ion a t m/e 658 showing that mouse blood largely contained ubiquinone-9 with a minor amount of ubiquinone-8 and a trace of ubiquinone-7. All of the molecular ions for the quinones were associat,ed with the molecular ions for t,heir corresponding hydroquinones. Determination of Ubiquinones in Parasitized Blood.-Frozen samples of Rhesus monkey blood infected with P. knowlesi (250 ml, 50%; parasitemia), Rhesus monkey blood infected with P. cynomolgi (425 ml, 1 3 C C parasit,eniia), and Swiss mouse blood infected with P. brrghri (273 ml, 70y0 parasitemia) were allowed to thaw at room t,emperature and were saponified by the same procedures used for the normal blood samples. The ubiquinone fractions, 5.0 mg, from Rhesus monkey blood infected with P. knowlesi, 3.5 mg from Rhesus monkey blood infected with P . cynomolgi, and 1.2 mg from Swiss mouse blood infected with P. berghsi, which were obtained in t,he same manner as the normal blood, gave a total ubiquinone content of 0.62 pgiml for blood containing P. knowlesi, 0.45 pg/ml for blood containing P. cynomolgi, and 1.03 pg/ml for blood containing P . berghei by the modified Craven's assay. Reversed-phase paper chromatography of the purified ubiquinone on Whatman S o . 3MRI paper impregnated with D o n Corning 550 silicone oil (5% in CHCI,) iising n-PrOH-H20 (7 :3, v: v), showed the presenceof ubiquinone10 and a lesser amount of ubiquinone-8 (Rr 0.32) for blood infected with P . knowlesi and P . cynomolgi, and ubiquinone-9 arid a lesser amount of ubiquinone-8 for blood infected with P. berghei. The mass spectrum of the purified ubiquinone obtained from blood infected with P. know/& and P. cynomolgi, determined at a probe temperature of 380", showed a molecular ion at m/e 726 for ubiquinone-8 and ions a t m/e 794 and m l e 862 for ubiquinones9 and -10. The mass spectrum of the purified ubiquinone obtained from mouse blood infected with P. berghei showed ions for ubiquinones-7, -8, and -9. All of the molecular ions for the quinones were associated with the molecular ions for their hydroquinones. Search for the Presence of Vitamin K in Normal and Parasitized Blood. A. Method of Lester and Ramasarma.8 Fresh samples of blood (120 ml) of the normal Rhesus monkey and Swiss mouse were lyophilized and exhaustively extracted, under subdued light, for 10 hr with EtOH-Et20 (1:3, v : v ) (1 1.). After filtrat,ion and evaporation of the solvents, the lipid residue was fractionated by preparative tlc, on silica gel G (20 X 20 cm plate; 1-mm layer) and developed 3 times in 3yc EtlO-hexane and once in l5TC EtzO-hexane. The region for K vitamins, det,ermined by reference samples of vitamin Kl(zo, and vitamins KZ(3j)and K?cro),was scraped from the thin-layer plate and eluted with ether. The residue, obtained after evaporation of EtzO, was sitbjected to reversed-phase paper chromatography as for residues containing the ubiquinones. The paper was developed with n-PrOH-HsO (85: 1.5, v:v). 90K vit,amins could be detected in either the Rhesus monkey or mouse blood by t'he method of Lester and Ramasarma.8 If any K vitamins were present the level would have been less t,han about, 9.0 pg, the limit' of sensitivity for this reagent. B. The Irreverre-Sullivan As~ay.~-The lipid fraction from fresh samples of lyophilized Rhesus monkey and mouse blood (120 ml each) was fractionated for K vitamins, as above, and the residue wm assayed for K vitamins by the Irreverre-Sullivan (7) R. F. Muraca, J. S. Whittick, G. D . Daves, Jr., P. Friis, and K. Folkers, J . Amer. Chem. Soc., 89, 1505 (1967). ( 8 ) R . L. Lester and T. Ramasarma, J . B i d . Chem., 284, 672 (1959). (9) F. Irreverre 4ncl M. IC. Sullivan, Sczenre, 94, 497 (1941).
,Joitrnnl oJ Merlicinul C h m i s t r y , 1970, Vol. I S , S o .
6 603
method9 as follows. Each residue was dissolved in 0.2 ml of abs EtOH containing 0.05 ml of a fresh 5co xolut.ion of sodium diethyl dithiocarbonate in abs EtOH (w:v); t'hen, 0.0.5 ml of S a O E t (1 g of Pl'a in 50 ml of abs EtOH) was added. The absorption of light a t 575 mp was followed over a 10-min period by comparing extiriction coefficients. Standard samples of meiiaquinone-9, as low as 4 pg, could be detected by this method. This method showed no change in E,,, values for the residues when compared with the E,,, values obtained by the measurement, of standard solutions of menaquinone-9. Therefore, no K vitamins could be detected in the residues from Rhesus monkey and mouse blood. The lipid residues from fresh samples (120 ml) of Rhesus blood infected with P. knowlesi (joyoparasitemia) and P. cynomolgi (13% parasitemia) and mouse blood infected with P. berghei (70%;parasitemia) were fractionated for K vitamins, as above. The method of Lest,er8and the Irreverre-Sullivang assay indicated no K vitamins could be detected in the parasitized bloods. C. Mass Spectral Analysis.-Samples of menaquinone-9 of known weight, were chromatographed by tlc; developed 3 times with 37, Et,*O-hexane and once wit,h l57C EtlO-hexane. The best method for introducing the sample into t,he mass spectrometer in order to obt,ain maximum sensitivity was to pack the silica gel, holding t,he menaquinone-9 sample, loosely in a probe designed to accommodat,e different amounts of silica gel. Det,erminations were made a t a probe temperature of 160". It' was possible to correlate the base peak oxonium ion fragment ( m / e intensity with the sample size by the use of this method. Samples of vitamin K, (2.0, 4 5 , 9.0, 18.0, and 30.0 pg) were added to Rhesus monkey blood (10 ml) and again isolated and chromat,ographed as above. The vitamin K1-fractionated residue was not isolat,ed from the silica gel. Mass spectral analysis of these vitamins Kl-silica gel mixtures, using the above method, gave a reproducible standard curve for the oxonium ion fragment ( m / e 2 2 5 ) intensities. The K vitamins fractionated lipid residues obtained from fresh samples (120 ml each) of normal Rhesus monkey blood and normal mouse blood were subjected to mass spectral analysis. The determinations were made at probe temperatures from 1!50 to 180". No base peak oxonium (pyrylium) ion fragment ( m 'c 225) could be observed in the samples. The K vitamins fractionated lipid residues from Rhesus monkey cted wit,h P. knowlrsi (35% parasit,emia) parasitemia) and mouse blood (150 ml) 70% parasitemia) were subjected to mass spectral analysis as above. S o oxonium ion fragment could be observed in any of the samples. D. [14C]Shikimic Acid Incorporation Studies.-[ W]Shikimic acid (uniformly labeled, 2.5 X l o 4 cpmlculture) was added to synt,hetic medium,lO and 6 duplicate in vitro suspensions (1.5 ml of blood cells in 9 ml of medium) of normal Rhesus monkey blood cells" and those infected with P . knowlesi were incubated for 8 hr at 3 7 O . 1 2 The incorporation of [14C]shikimic acid was measured under conditions designed for the intracellular in vitro cultivation of malarial parasites.'a The synthetic medium was based upon that described by Anfinsen, et a1.,l4 with modifications according 1 ~ acid was used to replace p l a ~ m a . 1 ~ to Geiman, et ~ ~ 1 . Stearic Bft,er incubation, the blood cultures were centrifuged, and the normal cells were combined as were the infected cells. The cells were washed t,wice with modified Ringer's solut,ion,16lyophilized, and exhaustively extracted with hexane as described by Skelton, d ~ 1 . 1 2 The residue, obtained upon evaporation of t,he hexane extrnrts, was suhjerted to tlr on silica gel G, and developed 3 (10) Q . X I . Geiman, K.A. Siddiqui, and J. V. Schnell, Mil. Med., 131, 1015 (1866). (11) T h e blood samples were drawn from uninfected Rhesus monkeys (Macaca mulatta) and Rhesus monkeys infected with P. k n o r l e s i , which was
originally isolated from a monkey (M.irus) from Malaya, and maintained by weekly intravenous inoculation of erythrocytic stages into Rhesus monkeys. Cultures were maintained under bacterial-free conditions. (12) F. S. Skelton, K. D . Lunan, K. Folkers, J. V. Schnell, IT,A. Siddiqui, and Q. 41. Gieman. Biochemistry, 8, 1284 (1969). (13) Q. M. Geiman, C. B. Anfinsen, R. W ,McKee, R . -4, Ormsbee, and E. G. Ball, J . Exp. Med., 84, 583 (1946). (14) C. B. Anfinsen, Q . M. Geiman, R. W.McKee, R. A . Ormsbee. a n d E. G . Ball, ibid., 84, 607 (1946). (15) W..1.Siddiqui, J. V. Schnell, and Q . M. Geiman, Science, 166, 1623 (1967). (16) Composition of modified Ringer's solution iisod for washing blood celh: XaCI. 8.21 g ; KCI, 0.30 g; CaCl?, 0.20y ; MgCI?, 0.10 p; and HtO. 11.
IO -
A.
8-
h
5 v,
-
'2
6-
2
4-
? 2
-
-_---- A00 -----__ ---_-X
2 -
p:
- -----
I-I 1
--7
I
I I I I
I I
2-
I I
j
726(COQa)
I 1
794 ( C O Ill I
600
640
b80
720
760
800
9,)
I I
1
a 40
880
m/e
time. with 3cb EtrO-hexnrie and once x i t h 155/cEtrO--hesane 83 described above. The K vitamins region, determined by vitamin Ki, K2(36),and K,c4a, references, was eluted with Et20 and the residue subjected to scintillation c o ~ n t i n g . ' ~X o radioactive material and, therefore, no K vitamins could be detected in either the iiormal or infected blood samples.
Discussion Ubiquinone-10 (111, n = 10) has been identified by reversed-phase paper chromatography (Rf 0.15) and ubiquinones-10, -9, and -8 (111, n = 9 and 8 ) have been identified by high-resolution mass spectrometry in normal Rhesus monkey blood. Ubiquinone-9 (reversed-phase paper chromatography, Ri 0.23) and ubiquinones-9, -S, and possibly -7 (111, n = 7 ) (high(17) Using a Nuclear-Chicago hquid scintillation spectroptiotometei
resolution I ~ : L * Sqrctromctry) were identified i l l t i o t w d mouse blood. The corresponding ubiquinones n-ere identified in Rhesus monkey blood infected with P. krioiclesi aiicl P. cynomolgi arid ill nioube blood infected witli I'. Oerghei but more ubiquinorie-S was found as evidenced by the appearance of ubiquinone-S in the reversedphase paper chromatograms of the ubiquinone-fractionated lipid residues from the infected Rhesus and mouse blood. l l a s s spectral results also indicate that the molecular iori for the more volatile ( i e . , ubiquinone7 > S > 9> 10) ubiquinone-S, obtained from the infected bloods, was of higher intensity than that observed for the corresponding ubiyuinone-8, obtained from normal bloods (Table I arid Figures 1 and 2 ) . Ubiquiriorie-7 was observed a b :t \I (d< but well-defined molecular ion
x 200 -----___ ___-_-----
re--I
I I I I
794
(c0 9,)
-------I I I 1
I
I I I
680
640
10
I
720
760
840
800
880
m/e
6.
0-
1
5 v,
2 3
__----x 200 __ -----____
6-
-
I
794 ( C O 9,)
r------I I I
----I
II
I / I
600
640
680
720
76 0
800
m /e Figure 2.-Xass
spectra (high-mass region) of ubiquinones (CoQ) isolated from (A) normal and (B) infected ( P . berghei) mouse blood.
in the mass spectrum of ubiquinone-fractionated lipid residues obtained from mouse blood infected with P. berghei. Only a slight trace of ubiquinone-7 was observed in the mass spectrum of the residue from normal mouse blood. These results are in accord with the finding of ubiquinones-8 and -9 in duck blood infected with P. lophurae. The same 14C-labeled ubiquinones were isolated from Rhesus monkey blood cells infected with P. knowlesi and P. falciparum incubated with [14C]-pHOC8H&O2H in synthetic medium. 12,18 The universality of distribution of ubiquinones-8 and -9 among various species of Plasmodium is supported by these results.
It is interesting to note that ubiquinone-7 was detected (mass spectral analysis) in mouse blood infected with P. berghei. This ubiquinone has also been demonstrated in rat liver by Linn, et aL16and Lawson, et ~ 1 . ' ~ The recently reported study of the in zdro incorporation of [ 14C]-p-hydroxybenzoic acid into ubiquinones in cultures of Rhesus monkey blood infected with P. knowlesi'2 did not show, unequivocally, that ubiquinone-7 is present in P. knowlesi. The finding of ubiquinone-7 in mouse blood infected with P. berghei may be exogenous to the parasite and endemic to liver tissue. The finding, by mass spectral analysis, of ubiquinones-10, -9, and -8 in normal Rhesus blood, and ubiquinones-9, -8, and possibly -7 in normal mouse blood,
(18) J. V. Sohnell, W. A. Siddiqui, Q. M. Geiman. F. 8. Skelton, K. D. Lunan. and K. Folkers, Biochemistry. 9, (in press) (1970).
(19) D. E. M. Lawaon, E. I. Mercer, J. Glover, qqd 9.. A. J l o r t o n , Bioclrem. J., 74, 38P (1960).
1'13
I' 13
i r i contrast to the finding of only ubiquinone-10 iii normal duck blood. Since thehe differential analysez :iw performed 011 the total ubiquinone content of n hole blood, any ubiquinone from hodj- tiisues v hich is tleliosited in the blood reservoir nil1 contribute to tlie ume that some of t'nc1.e multiple ubiquinone. may be blood coritamin:tnt~from tlie ubiquinones endemic to various tiisuri of the body Since t h e lilichu~:itid mouse bloods, infected \\ itli malarialpxra.ites. corit :bin t he s:tm(i mi :LY for the normal blood., it n:i. n c ~ relative degree of molecular iori i ubiquinone. in the ma^ -pectra. attenipt t o 4ion, in Table I :ind Figure. 1 :ind 2%t lie rnolecular ion ititeniitieu observed in tlie ma-:: spectw of tlie ubiquinone-fractionnted reciduci obt aiticd from t lie nialarin infected t h o d . The re.ult- clearlv indic:ttc the domin:mce of ubiyuinonc4 in t h e ma\s w t r u of tlie rwidues from the infected blood t i n c t ~ i r ti agreement TF it11 the predomi tiuiice of 14('-l:ibeledubiquinone-h obtained from in I ilro cdtiires of P. liiiowlehi n r i d 1'. jakiparccuc iilcubatcd i n -> iithetic medium cr)tit:iiriitig [14C']-p-h1droxybeiizoic acid. I h llircli time \\:is t a l w r i :mcl vfiort gii'en i i i .c:ircli ol I< vitamiiiq in norrn:~l aiici irifcct cd bl The chcniic:il tests, :iYderelopcd 1); Irreverrc nrid ;.lulliv:iiiIYTI ei-v tiegitivc fo as tis found for I< vitamin. fr:ictionated residues from normal ant1 infected duck blood high rezolutioii mu.s spvctrxl method and n i4C-labelir1g method n ere then perfected for detecting mere trace> of I< vit i i m i n b . Before K vitamim frnctioiintcd residue. from blood e submitted to liigli re-olution mass specsample. of' blood n ere "spiked" n it11knon n :inmullti of vitamin IC1 :itid the t1e.t method \\as 1.
