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Refe rence : Biol . Bu ll . 175: 154-166 . (Augus t, 1988 )
Inorganic A spects of the B lood Chem istry of A scidians.
Ionic C om position, and Ti, V , and Fe in the Blood
Plasma ofPyu ra chilensis and A scidia dispar
DOMINGO A. ROMAN, JUSTA MOLINA, AND LIDIA RIVERA
Departamento de QuÃmica,Facu ltad de C ienc ia s Bás icas ,Campus Co loso ,
Un iv er sid ad d e Ant ofa ga sta , C as illa 1240 Anto fa ga sta , C hile
A bstract. Iron, titanium , and vanadium analysis w ere
perform ed on the tunicates Pyura chilensis M olina,
1 78 2, a nd A sc id ia d isp ar, a nd th e in org an ic c hemistry o f
b lood was invest ig at ed . The ma jo r i on ic cha ract er izat ion
of the blood plasm a and cyto solic solutio ns w ere deter
m in ed . G el c hroma to gra ph y w as u se d to se cu re in fo rm a
tion on the possible existence of metal organic com
plexes.
P yura chilensis accumulates F e and Ti, and A scidia
dispar accumulates Fe, Ti, and V in blood cells in this
quant ita tiv e o rd er. S ig nific an t le ve ls o f me ta ls a re a ss oc i
ated w ith cell residues (m em brane c ells), although this
m ay be, to som e extent, dependent o n the cell lysis tech
nique.
T he elu tion behavio r of plasm a in S ephadex G -75 and
L H-20 gels an d the respective absorption spectra of the
fra ctio ns s howed evid en ce o f o rg an ic me ta l c omp le xe s in
th e p la sm a o f b oth tu nic ate sp ec ie s.
Introduction
For y ea rs tu nic ate s h av e p iq ue d th e c urio sity o f b io lo
g is ts b ecau se o f t he ir unu sual phys io logi ca l p ecul ia ri ti es
and because they m ay have given rise to the vertebrate s
(Be rr il , 1955) . Among the phy siolog ical p ecul ia ri ti es t ha t
distinguish these organ ism s from othe rs are the follow
ing: (i) T hey need a low ten sion o f oxy gen (G oodbo dy,
1 97 4). T o d ate , n o re ve rsib le b in din g o f o xy ge n h as b ee n
detected nor the unequivocal existence of a proteic O 2
transport compound that transports O2 through the
blood (M acara et al, 1979a; Agudelo et al., 1982). (ii)
T hey are en tirely am m onotelic in their protein m etab o-
Rec eiv ed 6 J ul y 1 98 7; a cc ep te d 2 0 Ma y 1 98 8.
lism , but are uricotelic w ith respect to nucleic acid m e
ta bo lism. The re fo re , t he y d iffe r from mo st in ve rte br ate s
that are w holly am monotelic, accum ulating uric acid
and purines in nephrocyte vacuoles (G oodbody, 1974;
Wr ig ht, 1 98 1). T he fu nc tio na l importa nc e o f th is s to ra ge
rem ain s obsc ure, (iii) T hey are cap able of h um oral and
c ell ula r immunolo gic al re sp on se s (Wr ig ht , 1 98 1) a nd a re
rich in bio -active substances (R om an, 198 6). (iv) T hey
a cc umula te m eta l io ns.
W ith re sp ec t to m eta l io ns, tu nic ate s a re k nown fo r th e
u pta ke o f se le cte d m eta ls from se aw ate r a nd fo r a cc umu
lating the m in their blood (C arlisle, 19 68; S winehart et
ai, 1974; Senozan, 1974; Biggs and Sw inehart, 1976).
M em bers of the ord er E ntero gona can acc um ulate vana
dium (Kustin et al., 1975; Kustin and M cLeod, 1977;
M acara et a l., 19 79b; B igg s a nd S win ehart, 197 9; B otte
et al, 1979; Dingley et al, 1981; H ori and M ichibata,
1 98 1 ; R owle y, 1 98 2; D in gle y e t a l, 1 98 2). H ow ev er, th e
type of co ordination co mpound(s) in w hich th e m etal is
involved in the blood is unknow n (C arlson, 1975; T ul-
lius et al, 1980; Dingley et al, 1982; Hawkins et al,
1983a; B ruening et al, 1985; Frank et al, 1986). M em
bers o f th e o rd er P le uro go na , s ub -o rd er S to li do bra nchi-
ata, accum ulate iron (Endean, 1955a, b, c; A gudelo et
al, 1982; Agudelo et al, 1983a, b; Agudelo et al, 1985),
but nothing is known about its function in blood cells
(Hawkins et al, 1983b). In plasm a, iron is associated
with transferrin-like m etalloproteins (M artin et al,
1 98 4; F in ch a nd H ue be rs, 1 98 6).
H aw kins et al (198 3c) proposed that ascidian taxon
om y reflects a separation into van adium - and iron-con
taining species. T un icates accum ulate other m etals be
sides vanadiu m and iron (M onniot, 1978; M acara et al,
1 979c; A gudelo e t al, 1 981; R ow ley, 1982), w hich m ay
15 4
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INORGANIC ASPECTS OF THE BLOOD CHEMISTRY OF ASCIDIANS 155
n ot b e e sse ntia l e leme nts su bje cte d to se le ctiv e a cc umu
la tio n mech an ism s. S es sile fil te r fe ed in g anima ls a re v ery
sensitive to their im mediate environm ent, and signifi
cant am ounts of contam inating m eta llic e lem ents could
be taken up by ascidians (Papadopoulou and Kanias,
1977).
In p ro ce sse s in w hic h m eta ls a re a cc umula te d in b lo od
cells, it is logical tha t m etals m ake a transient or perm a
nent appearance in blood plasm a. O nce m etals gain ac
cess to the body interior, they m ust be app ropriately dis
tribute d, bu t beca use of its hydrolysis property som e of
these m etals cannot be held in solution, in the interior
m edia, w ithout som e m echanism to prevent its precip i
tation.
N o m etalloproteins such as hem ocyanin h ave been re
ported in ascidian blood plasm a. H ow ever, H aw kins et
al. (1980a) and W ebb and Chrystal (1981) studied the
meta l b inding p rope rt ie s—inc luding spect ra l cha ract er
iz atio n and me ta l c on te nt sâ €”of s ome t un ic ate s (H awk in s
et a i, 1980 b). They fou nd p relim inary evidence of m etal
com plexing. T his w as co nfirm ed by M artin et al. (198 4)
in the plasm a ofP yura stolonifera, by d em onstrating an
iro n-b in din g p ro te in o f a bo ut 4 0,0 00 d alto ns mole cu la r
w eigh t w ith one iron-bin ding site considered as one P y-
u ra tr an sfe rr in ( Fin ch and Hue be rs , 1 986).
In this work, the Ti, V , and Fe contents were deter
m ined in s ever al t is su es. A lso, t he ma jo r cha ract er izat ion
and C hrom atograp hie elutive behavior on S ephade x G -
75 and L H-20 gels of P yura chilensis M olina, 1782 , and
A scidia dispar blood plasm a w ere exam ined. T hese are
two phy logene ti ca ll y d iv er se a sc id ians .
Material s and Methods
Chemic als w ere from Merc k. 3 ,3 '-d im eth yln ap hth i-
d in e w as from E astm an o rg an ic c hemic als a nd o ph en an -
th ro lin e h yd ro ch lo rid e w as from R ie de l-D e H ae n. S ep h
ad ex G -75, L H-20 gels and b lue dextran 2000 w ere from
Pharm acia Fine C hem icals. D eionized w ater w as pre
pared from distilled w ater passed throu gh a disposable
demine ra li ze r ca rt ri dge (Co rn ing 3508-B).
S pe cim en s o f P . c hile nsis a nd A . d isp ar w ere c olle cte d
a t B ahÃaMejillo ne s d el S ur (A nto fa ga sta -C hile ) from
m arine pools, in w hich they w ere found as encrusting
fo ulin g o rg an isms . P . c hile ns is a fix es its elf t o ro pe s while
A . d isp ar a tta ch es itself to p ain ted flo atin g m eta llic b ar
re ls w he re th ey c oe xis t w ith h yd ro zo an s a nd b ry oz oa ns.
B efore draw ing blood, specim ens w ere m aintained for
som e tim e in seawater at room tem perature, and then
w ere g en tly sq ue ez ed to remo ve most o f th e se aw ate r.
B lood sam ples of both species w ere obtained by cut
ting the base o f t he bo dy. B lood cells w ere rem oved from
the plasm a by centrifuging (2 500 rpm ; 1 0 m in). P la sm a
w as k ep t a t 4 -5°Cw hile c arrie d to th e la bo ra to ry a nd w as
u se d a s so on a s p ossib le .
C ellular resid ues presum ably consisted of cell m em
branes. N o distinction w as m ade betw een cell surface
and intracellula r m em branes. C ell sam ples w ere rinsed
w ith seaw ate r and then subjecte d to tw o diffe rent c ell ly
s is p ro ce sse s. In th e first p ro ce du re , c ells w ere su bje cte d
to th re e fre ez e-th aw c yc le s in d eio niz ed w ate r m ed ia ( 1 .4
parts of triturated ice + 2 parts of C aC l2 X 6H 2O freeze/
ro om temp era tu re ), g en tly sq ue ez ed w ith a c ell te flo n h o-
m ogenizer, and then centrifugated at 8000 rpm . In the
seco nd procedure cells w ere su bjected tw ofold to an ex
cess of m ethanolic solution of 0.75% HC1 (Hawkins,
pers. c om m.) and centrifuged at 800 0 rpm . In both cases
th e c yto so lic so lu tio n a nd m eth an olic e xtra ct w ere m ad e
up to the original volum e from w hich the cells w ere ob
tained. W hole blood sam ples of P. chilensis were sub
je cte d to th e firs t c ell ly sis p ro ce du re , b ut w ith ou t d eio n
iz ed wate r. A Sorv all re fr ig era te d c en trifu ge was u sed.
Meta l ana ly si s
P rior to the T i, V , and F e de term inatio ns in specim en s
and tissues, a q ualitative analysis w as perform ed on di
g es te d b lo od c ells . C el ls r in se d w ith m ic ro filte re d s eawa
te r w ere d ig este d w ith b in ary H NO 3/H C1O4 a cid sy stem
(Jones et al., 1982), perform ing assays for C u, M n, Fe,
Ni, C o, Ti, V , and Nb (Feigl and A nger, 1972). M n and
F e w ere also subjec ted to sem i-quantita tive assays w ith
Me rk oquant s tic ks .
Pyu ra c hile ns is a nd A sc id ia d is pa r w er e ana ly ze d in di
vidually . Tissues including blood w ere obtained from
10-20 specim ens of P . ch ilensis and 30-5 0 sp ecim ens of
A . d ispar. B odies w ere separated from tu nics and rinsed
w ith filte re d se aw ate r. T un ic s w ere g en tly sc ru bb ed w ith
a plastic brush to remove dirt and rinsed in a sim ilar
manner. S iphons and tunics were cut off w ith a hard
acrylic knife. Specim ens and tissues, including som e
samp le s o f p la sm a, c ells, a nd c ellu la r re sid ue s, w ere th en
d rie d a t 1 10°Co c on sta nt w eig ht, d ig este d w ith a b in ary
acid procedure (Jones et al., 1982), and then treated ac
cord in g to th e re sp ec tiv e me ta l a na ly sis .
In tis su es , iro n wa s d ete rm in ed w ith 1 ,1 0-p henanth ro -
lin e (S an de ll, 1 95 9; F rie s, 1 97 2), a nd T i a nd V w ere se pa
rated (Korkisch, 1969; Fukasawa and Yam ane, 1977)
p rio r to th eir d ete rm in atio ns. T ita nium w as d ete rm in ed
according to Q ureshi et al. (1968), and vanadium using
the m ethods of Bannard and Burton (1968) and Fuka
saw a and Y am ane (1977). In the fractions, iron w as de
t erm ined u sing 2 ,4 ,6 -t ri -2 pyr idy l- l, 3, 5- tr ia zi ne (Col li ns
et ai, 1959; Box, 1981), and vanadium and titanium as
above, without separating them after digestion of the
fra ctio ns w ith a b in ary H NO 3/H C1O4 a cid sy stem (Jo ne s
et ai, 1982).
B lan k con trols w ere used in e very m etal an alysis, an d
except in the fractio ns, all the determ in ations w ere per
f ormed in t ri pl ic at e.
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156
D. A. ROMAN ET AL.
Dete rm inatio n o f th e majo r io nic c ompo sitio n
and relat iv e reduced f ea tu re o f the f l uids
Chlo rin it y a nd s alin ity w e re d ete rm in ed con du ctime t-
ric ally w ith re sp ec t to sta nd ard s eawa te r a t 2 5Â °C(c on -
ductim eter Radiom eter CD M 2e, with a standard cell
C DC 1 04). C hlorid e w as de term ined by M ohr titration
a nd su lp ha te b y d ire ct titra tio n w ith b arium p erc hlo ra te
u sin g Tho rin a s in dic ato r. S ub sequ en tly , c atio ns w er e re
m oved by passing the sam ple through a strong acid cat
ion exchange resin column (Fritz and Yamamura,
1 95 5), e xc ep t in se aw ate r in w hic h c ase s ulp ha te w as d e
te rm in ed g ra vime tric ally a s BaSO4. Suc ce ss iv e d et erm i
nation of calcium and m agnesium w ere m ade by poten-
tiometric titra tio n w ith a c alc ium io n s ele ctiv e e le ctro de
(Rom an et al, 1982); Na, K ., and Li analysis w ere per
fo rm ed b y flame emissio n sp ec tro ph otome try o n a R ad i
om eter F LM -3; p H m easurem ents w ere m ade poten tio-
matrically on a Radiometer pH M eter 26 with glass
m embra ne e le ctro de . A ll m ajo r c ompo ne nt d ete rm in a
tio ns w ere made in trip lic ate .
The relative reduced feature of the plasm a and cyto-
so lic so lu tio ns w ere te ste d b y tw o re do x p ote ntiome tric
titrations (non-stan dard bioche mical m ethods). In the
firs t, a liq uo t s amp le s (1 0-2 0 m l) in p ol yp ro py le ne v es se l
w ere pu t into a R adiom eter T TA -80 titratio n assem bly,
acidified w ith 0.75% H C1, and then titrated w ith a stan
d ard so lu tio n 0 .1 7v ~K MnO 4. In th e se co nd , a liq uo t sam
p le s (1 0-2 0 m l) w ere a cid ifie d w ith 2 m l o f c on ce ntra te d
HC1O4, treated with 5 m l of a standard solution 0.1 N
K .2C r2O 7,and then titrated w ith a standard solution o f
Fe(II).
Chromatographie f ract iona tion o f b lood p la sma
F ra ctio na tio ns w ere p erfo rm ed o n S ep ha de x G -7 5 a nd
L H-2 0 ge ls, in therm ostated chrom ato graphy colum ns
(P harm acia Fine C hem icals K 26/40) loaded w ith 4 g of
Sephadex G-75 and 13 g of Sephadex LH-20, respec
tiv ely. T he colum n tem pe rature w as 20Â °C ,but all sam
p le s a nd e lu an ts w ere c oo le d a t 4 -5°C .P la sm a samp le s
w ere c on ce ntra te d b y fre ez e-d ry (F re ez er-d ry er-5 L ab -
c on co ), fiv e-fo ld fo r P . c hile nsis a nd tw o-fo ld fo r A . d is
par b efo re r unnin g th e c hr omatogr aphy p ro ce du re s. T he
void volum e of the colum n (V 0) w as determ ined using
blue dextran-2000, and the bed volum e (V t) w as calcu
lated according to the height and diameter of the gel
column.
In G -75 chrom atography the sam ple volum es w ere 6
an d 10 m l for A . dispar and P . chilensis; the elu ants w ere
0 .0 1 M N aC l a nd 0 .0 6 M a ce tic a cid , re sp ec tiv ely , c oo le d
an d dea ereated, collec ting fractions of 10 m l (plasm a o f
A. dispar) and 6 m l (plasm a of P. chilensis). The absor-
bance at 278, 288, 310, 375, 454, and 675 nm (plasm a
of P. chilensis), 265 and 322 nm (plasm a of A. dispar),
and the m etal analysis in all fractions w ere m onitored
with respect to eluant solutions, previously passed
th ro ug h th e re sp ec tiv e c olumn , a s re fe re nc e o r b la nk , re
spectively.
In L H-2 0 c hroma to gra ph y, to m in im iz e in homo ge ne -
ities in the colum n the gel w as packed after sw elling in
deaereated m ethanol. O ne bed volum e colum n of each
o f t he fo llowin g e lu an ts: w ate r, 2 5,5 0 a nd 7 5% meth an ol
in w ate r (v/v), w ere then passed through th e colum n, fol
lowe d b y 9 9.8% meth an ol, c olle ctin g tw o 1 0m l fra ctio ns
per eluant for use as reference or blank solutions. T he
colum n w as then loaded w ith the sam ple (10 m l). C hro
m ato gra ph y w as p erfo rm ed u sin g 1 .5 b e d v olume o f e ac h
cooled deaereated m ethanol/w ater gradient from 0 to
99.8% m etha nol acc ording to M acara et al. ( 1 979b), col
le ctin g 6 m l (p la sm a c hroma to gra ph y o f P . c hile ns is) a nd
10 m l (plasm a chrom atography of A . dispar) fractions.
A bsorba nce at 272, 288, 3 10, 32 0, 375 ,454, an d 675 nm
(plasma of P. chilensis), 266, 280, 326 and 660 nm
(p la sm a o f A . d isp ar), a nd m eta l a na ly sis in a ll fra ctio ns
w ere mon ito re d. U ltra -v io le t a nd v isib le sp ec tra w ere re
c ord ed fo r w ho le p la sm a a nd th e p ea k-fra ctio ns from th e
e lu tin g p atte rn s, emp lo yin g a B ec km an 3 5 sp ec tro ph o-
tom eter. A ll oth er ab sorciom etric m easurem ents w ere
a ls o made u sin g th is in strumen t.
Results
Meta l ana ly si s
Me ta l c on ce ntra tio ns fo un d o n sp ec im en s a nd v ario us
tissu es o f tu nic ate s a re liste d a s mg/K g d ry w eig ht (T ab le
I). C on centration s for plasm a are given in m g/1. N i, C o,
M n, and N b w ere not detected in blood cells. H igher con
c en tra tio ns o f iro n a nd tita nium, a nd iro n, tita nium, a nd
vanadium w ere found in P . chilensis and A . dispar blood
c ells, re sp ec tiv ely . A lth ou gh V w as n ot d ete cte d in P . c hi
lensis blood cells a nd w as found in A . dispar blood cells,
only trace lev els of it w ere fou nd in bo th bloo d plasm as.
T i w as n ot d ete cte d in P . c hile nsis b lo od p la sm a.
R esu lts o f the m etal analysis in cell lysate (c ytosolic
s olu ti on ), c al cu la te d b y th e d iffe re nc e b etw een th e me ta l
conten ts in w hole blood cells an d in blood cell residues,
are tabulated as percentage of m etals in Tables Ila, b, c,
re sp ec tiv ely . H e re , th e c ellu la r re sid ue s w ere n ot w as hed
with acid prior to analysis. Aqueous and 0.75% HC1/
m ethano l cell lysis procedures w ere c onsidered (P . chi
lensis). These resu lts show that iron conten t in cell resi
dues from aqueous and H Cl/m ethanol cell lysis proce
dures a re low and com parable, but the titanium co ntent,
surprisin gly, w as higher an d greater in the c ellular re si
d ue s th an in th e c yto so lic so lu tio n fo r b oth c ell ly sis p ro
c ed ure s, b ut h ig he r in c ell re sid ue s from H Cl/m eth an ol
lysis m ethod. T herefo re, it is possible that m etallic pre
c ip ita tio n b y exte ns iv e h yd ro ly sis (Agude lo e t a l. , 1 98 3b ,
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INORGANIC ASPECTS OF THE BLOOD CHEM ISTRY OF ASCIDIA NS
Tabl e I
Rela tiv e d is tr ib uti on o ff e, T i, a nd y c on te nts (mg /k g d ry ) i n P yu ra c hi le ns is a nd A sc id ia d is pa r
15 7
SpecimenBody
(withoutunic)SiphonsTunicBlood
plasmaBlood
cellsBlood
cellesidues' Blood
cell residues0Fe191.884.370.7243.945.0 (1.51,105.47.417.3P
. chilensisTin.d.n.d.n.d.n.d.n.d.n.d.277.8132.825 8.9Vn.d.n.d.n.d.1.51.90.06n.d.n.d.n.d.Fe9 4.1717.48
isparTi107.253.116.2125.661.42.11,5
amg /1 ; b fr om l ys ed c el l p re pa ra ti on s p ro du ce d b y s ub je cti ng th e c ell s ampl es t o s ev er al f re ez e- th aw c yc le s i n d eio niz ed w at er m ed ia a nd th en
c en tr if ug in g th em a t 8 00 0 r pm ; c fr om ly se d c ell p re pa ra ti on s p ro du ce d b y s ub je cti ng t he c el l s ampl es to t re atm en t w it h m eth an ol ic s ol ut io n o f
0 .7 5% HC1cen tr if ug in g t hem a t 8 000 rpm ; dno t d et erm in ed ; n .d . = not d et ec te d.
1985) m ay hav e been m inim ized d uring w ater c ell ly sis
in the conditions of this w ork . T hus, it appears that m ore
atten tion sho uld b e focused on tunicate blood c ell lysis
procedures.
Table I la
Rela t ive iron d is tr ibu tion in b lood ce ll s as de te rmined
in pooled samples
SpeciesP.
chilensisP.
chilensisA.
disparCytosolicd-csolution99.3 98.4 f73.1 Cellresidues0.7 1.6 '26.9
Table l ib
Rela t ive t itanium dis tr ibu tion in b lood ce ll s as de te rmined
in pooled samples
P. chilensis
P. chilensis
A. d ispar
52.2%
6.8%r
49.5%
47.8%
93.2%f
50.5%
Tabl e He
Rela tive vanad ium dis tr ibu tion in b lood ce ll s as de te rmined
in pooled samples
A. d ispar 76.4
23.6%
dCa l cu la te d by d if fe re nc e b etwe en con te nt s i n whol e b lo od c el ls a nd
i n b lo od c el ls r es id ue s (Tab le I ); e in r es pe ct t o l ys ed c el ls p re pa ra ti on s
p ro du ced by sub je ct in g t he c el l s amp le s t o s ev er al f re ez e- th aw cyc le s i n
d ei on iz ed w at er m ed ia /c en tr if ug in g th em a t 8 00 0 r pm ; fwi th r es pe ct
t o l ys ed c ell p re pa ra tio ns p ro du ce d b y s ub je cti ng t he c el l s ample s t o
t re atmen t w i th a me th anol ic s ol ut io n o f 0 .7 5% HCl/ ce nt ri fu gi ng t hem
a t 8 00 0 r pm .
Io n c ompos itio n and r educ ed te nd en cy o f b lo od flu id s
T he pH and ionic com position o f plasm a, lysed w hole
blood, and cytosolic solutions, for both species, are
shown in Tables III and IV. The sulphate content in A.
d ispar plasm a w as greater than in P . ch ilensis, b ut both
contents w ere low er than in seaw ater. In the cytosolic
s olu tio ns, e .g ., a qu eo us in tra ce llu la r m ed ia , th e c on ce n
trations of su lphate w ere lo w w ith resp ect to the plasm a.
C alcium and m agnesiu m c ontents in P . chilensis plasm a
are higher than in A . dispar. In P . chilen sis so me enrich
m ent occurred w ith respect to seaw ater, w hich also oc
curs for sodium and potassium . C alcium , m agnesium ,
sodium , and potassium contents also were lower in
w ho le ly sed blood tha n in plasm a (P . c hilensis). In P . chi
len sis b lo od ce lls, th e so dium co ncen tra tio n in th e cyto
so lic so lu tio n is o nly 5 0% o f th e A . d isp ar c yto so lic s olu
tio n. H ow ev er, p ota ssium c on ce ntra tio n is v ery low.
The pH of the whole lysed blood (P. chilensis) was
n ea rly a lk alin e, th e sa lin ity a lm os t e qu al to th e se aw ate r
from w hich the specim ens w ere obtaine d. T he sulp hate
c on ce ntra tio n w as o nly 6 2% o f i ts c on ce ntra tio n in b lo od
plasma.
P lasm as, 0.75 % H Cl/m ethanolic extracts from blood
cells, an d cy tosolic so lutions had redu cing tendency in
b oth s pe cie s in re sp ec t to d ic hromate and p ermang an ate ,
respectively.
Spec tra l- separative Chromatographie behav ior of i ron ,
tita nium, a nd va na dium in p la sm a
P. chilensis plasma is pink-orange and A. dispar
p la sma is g re en is h-y ellow . F ig ure 1 s h ow s th e U V -v is ib le
spectra of both species' plasm a. The bands 265-290,
300-330, and 675 nm regions were common to both
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158
D. A. ROMAN ET AL.
T ab le II I
I on ic c ompos it io n o f t he b lood p la sma o f Pyura chi le ns is
am/Asc id ia d i spa r
ChlorinityoSalinity
%oci-(g/i)SOI
(g/1)Ca2+
(g/1)Mg2+
(g/1)Na+
(g/1)K+(g/l)Li+
(mg/1)pHNa/KCa/MgPlasma
/ . chilensis19.3832.3519.860.600.511.5415.680.780.876.770.3320.10Plasma
A.dispar18.6433.6719.050.790.341.149.340.420.846.480.3022.20Surface
coastal
seawater 19.4435.1319.512.600.441.3311.160.401.328.030.2327.90
•urface coasta l seawater of BahÃaMejillones del Sur.
sp ec tra , w ith a lig ht b ath oc hromic e ffe ct in th e U V b an ds
of A . dispar plasm a w ith respect to the P. chilensis U V
spectrum of plasm a, w hich also show s a shoulder in the
280-290 nm zone.
The elution patterns detected at 265 nm for A . dispar
and at 310 nm for P. chilensis are given in Figure 2a.
N on e o f th e P . c hile nsis fra ctio ns w ere c olo re d, b ut fra c
tio ns 9-11 w ere yello wish in A . dispa r plasm a chrom a-
tography. T he elution p rofiles of iron and vanadiu m for
Table IV
Ionic compos it ion o flysed who le b lood and cy tosol ic so lu t ions
o f Pyura chi le ns is and Asc id ia d is pa r
ChlorinityaSalinity
%oor
(g/i)SO;
(g/1)Ca2+(g/l)Mg2+
(g/1)Na+(g/l)K+(mg/l)Li+(mg/l)pHNa/KCa/MgLysed
whole
b lo od o f
P. chilensis^19.7435.6619.570.370.301.039.59484.900.947.820.2919.8Cytosolic
o lu ti on o f
P. chilensis*n.m.n.m.n.m.0.070n.d.n.d.0.05111.20n.d.7.014.6—Cytosolic
o lu ti on o f
A. ispar*n.m.n.m.n.m.0.051n.m.n.m.0.1040.300.027.36346.7—
* A na ly sis o n lys ed w ho le b lo od o f A . d isp ar w ere n ot m ade d ue to
lack o fsamp les ; b from subjec ting the samples o f b lood to seve ral f reeze -
th aw cy cle s an d th en ce ntrifug in g a t 8 00 0 rp m; from su bjec tin g th e
ce ll s amp les to seve ral f reeze -thaw cycles wi th de ion ized wa ter and then
centrifuging them at 8000 rpm ; n.m . = not m easured; n.d. = not de
tected.
35
4
45
8
55
WAVEL.ENOTH IN RITI
32 0
650
36 0
75 0
Figure1 . Ul traviolet -v is ib le spectraof blood plasma ofAsc idia dis
p ar ( co nc en tr at ed twof ol d b y fr ee ze -d ry a nd a ci di l ia i a t pH 3 w ith a ce
ti c a ci d, th at a ls o w as th e re fe re nc e s olu tio n AUV†¢€¢€¢fresh , water as
refere nce A uv ,v is ), an d P yura ch ile ns is (fresh , w ate r as refe re nce
BUV.V ISo lid l in e) . C el l p ath le ng th 1 cm. D il uti on s hown were a pp li
cable.
P . c hilen sis, and iron, tita nium , and vana dium for A dis
par are also presented in F igure 2b. In both species pat
terns, two peaks were obtained with respect to absor-
bance, each one in fractions 3,4; 6-9 (A . d ispar), and 5,
6; 10-12 (P. chilensis). The first band eluted w as in the
void volum e of the colum n (V 0 = 30 m l) and should have
c ontained com poun ds w ith greate r m olecular w eight or
a t least co mparable to the u pper ex clusion lim it o f t he G -
75 colum n bed. The second band eluted w as at a greater
volum e than V ,(V e = 61 and 67 m l for P. chilensis and A .
d is pa r p la smas , r es pe ctiv ely ) a nd s hould hav e c on ta in ed
c ompo un ds w ith le ss m ole cu la r w eig ht o r c ompa ra ble to
the low est e xclusion lim it o f the G -7 5 co lum n bed. T his
a lso sh ou ld b e v alid fo r th e y ellow fra ctio ns (9 -1 1) from
A . d isp ar p la sm a ch roma to gra ph y. T he a bso rb an ce p ro
file a t 3 22 nm sh ow ed e qu al c ha ra cte ristic s fo r A . d isp ar,
and the sam e o ccurred w ith the profiles at 278,288, 375,
454, an d 6 75 nm for P . c hilensis.
F ou r p ea ks w ere o bta in ed fo r P . c hile nsis w ith re sp ec t
to th e iron con tent in fractio ns, w hose elution volum es
(V e) were 13,25,55, and 67 m l. The second peak had the
s ame v alu es o f th e Chromato gra ph ie b eh av io r p aramete r
(V e /V0, V e/V t, K av) of th e firs t b an d in fu nc tio n o f a bs or
bance at 31 0 nm , and so on. T hese fractions (5,6 ) sh ould
have contained iron compounds of high molecular
weight, found for the first iron band. The other peaks
should correspond to iron com pounds of low m olecular
w eig ht. V an ad ium w as a lso e lu te d a fte r th e b ed v olume .
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INORGANIC ASPECTS OF THE BLOOD CHEMISTRY OF ASCIDIANS 159
7 21 61
15
FRACTION
F ig ur e 2 a. E lu tio n p at te rn s o f b lo od p la sm a o fA s cid ia d is pa r f rom
Seph ad ex G-75 chr oma to gr ap hy a t 2 70 nm (6 m l c on cent ra te d two fo ld
b y free ze -dry ing , 6 m l frac tio ns , A d as h lin e), a nd P yu ra ch ile nsis at
3 10 nm ( 10 m l c on ce nt ra te d f iv efo ld b y f re ez e- dr yi ng , 6 m l f ra cti on s,
B so lid l ine ).
Four bands were also obtained for A . dispar with re
spect to iron content in fractions (V e = 57, 87, 107, and
137 m l). None had the same values of the Chromato
graphie param eters of the bands in function of absor
bance at 265 nm . The four V e values are greater than the
V t, therefore they should not contain iron com pounds
of high m olecular w eig ht. H ow ever, for tita nium (three
bands, Ve = 37, 87, and 117 ml) the first peak is super
posed and sim ilar in the profile at 2 65 nm , w hic h should
m ean that it corresponds to titanium com pounds w ith a
high m olecular w eig ht. T he o ther ba nds are after the bed
v olume . T he e lu te d v an ad ium sh ow in cre asin g c on te nts
after fraction 10, fo r w hich only tw o bands w ere consid
ered (Ve = 67 and 87 ml), both after the bed volume,
where the first is superposed with the second peak at
265 nm .
The elution profiles for P. chilensis and A. dispar
bloo d pla sm a chrom atography on S eph adex L H-20 , em
ploy ing m ethanol/w ater gradient as eluants are give n in
Figures 3 and 4. N one of the fractions w ere colored. A t
272 nm, two major bands and one shoulder were ob
tain ed for P . c hilensis, e ach in fraction s 6-9, 11-13, and
14-15. At 310 nm , three bands and two shoulders were
obtained, each in fractions 6-9, 11-12, 14-15, and 18-
19, respectively . P rofile s w ere also detected a t 288 nm
(which is superposed with the profile at 272 nm ), 320,
375 nm (w hich w ere superposed w ith the profile at 310
nm), and at 454, and 675 nm, which were superposed
betw een th em (no bands w ere obtain ed in frac tions 5-6,
7-8, 10-1 1, and 15-16).
10
FRACTION
15
20
F igure 2b. E lution patterns of m etal contents per fraction from
Sep ha de x G -7 5 c hr om at og ra ph y: in p la sm as o f P yw a c hil en si s ( ir on
E so lid lin e; va nad ium F ~), an d A sc id ia d isp ar (iron G o pe n circles;
titanium H ; vanadium I c losed circles). C o nditions, sam ples, and
f ra ct io n vol ume s a re o f F ig ur e 2 a.
In A. dispar, 6 peaks and I shoulder were obtained at
266 nm , each in fractions 4-6, 9, 15-16 (shoulder), 21-
22, 25, 29, and 34 (small). A t 326 nm one major band
w as obtain ed (fractions 3-6), a lthough tw o sm all peaks
w ere also observed at fractions 29 and 34, respectively .
In addition, patterns w ere detected at: 288 nm (that w as
not superposed with the profile at 266 nm , only for the
shoulder, fraction 11) and at 660 nm (no bands w ere ob
tain ed in fractions 2-3 an d 33-34).
Iron w as eluted in a ll L H-20 chrom atography of P . ch i
lensis plasma. The Ve of the main bands were at 49, 61,
85, 97, 115, 133, and 235 m l. The first three bands w ere
sup erposed w ith the re spective eluting p eaks at 272 nm ,
and also with three eluting bands of the profile at 310,
and w ith tw o peaks of eluting profile at 675 nm . M ost of
the m ain iron bands in the profiles were observed at a
grea ter volum e than V , o f the bed co lum n, and after frac
tion num ber 20, appeared not to have association w ith
the patterns at 272, 288, 310, 375,454, and 675 nm . V a
n ad ium w as n ot c on sid ere d in th is o pp ortu nity .
Iron was also found in all LH -20 chrom atography of
A. dispar, and the Ve of the m ain peaks were obtained at
35, 55 (shoulder), 115, 145, 165, 1 95, 21 5, 265 , 295, and
3 30 m l, in w hic h th e C hroma to gra ph ie p arame te rs o f a ny
of th em correlates w ith the eluting peaks w ith respect to
absorbance elutin g p atterns. T itanium w as not found in
fractions 7-14, and the Vc of the main bands were ob
tained at 45, 155, 185, 205, 265, and 305 m l. The second
titanium eluting band correlates with the respective
peaks in the profile at 266 nm, and the fourth is super
posed with the patterns at 266 and 280 nm . Vanadium
was found in all the chromatography, but most was
8/19/2019 Pyura chilensis - Heavy Metals
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16 0
D. A. ROMAN ET AL.
22.^3.8
l °°°°,.
0 00
0000
00„
° o
°0°oooo
oo0/V°oo0°0Oo°
°°°,
o
o
oo
oo
oo
o o
O o
o o
O o
o o
O o
o o
O o
o
o
o
o
o
o
=00°
o°V
o o
15 25
6 ml FRACTIONS
35
45
F ig ur e 3 . E lu ti on p at te rn s o f b lo od p la sm a o fPyu ra c hil en si s f rom Sep ha de x LH -2 0 c hr om ato gr ap hy
at 2 72 n m (C so lid lin e) a nd 3 10 n m (D X X X ), an d e lu tio n p ro file o f iron (c irc le s). 1 0 m l co nc en tra te d
f ive fo ld by f reeze -drying, 6 ml f ract ions.
eluted from fractions 1-17 (V e = 5, 25, 75, 95, 115, 145,
265, 285, and 335 m l).
F ractions 5 and 11 absorp tion spectra fro m S ephadex
G -75 chrom atography of P. chilensis blood plasm a are
show n in F igure 5a. F ra ction 5, th at also correspo nds to
the second iron-band in the respective eluting profile
(Fig. 2b), had an absorption band at 276 nm with one
should er a t 400-42 5 nm . F raction 11 show s absorption
m axim a at 270, 310, and 460 nm w ith a shoulder at 360-
375 nm , and is not in the area of an iron-band, although
it is betw een the third and fourth iron-band, in the re
s pe ctiv e e lu tin g p att ern (F ig . 2 b).
2.1
rHO|55
15
FRACTION
A.ayw
0
^
i
a
o
H
5
z
O
25
35
F ig ur e 4 . E lu tio n p att er ns o f b lo od p la sm a o fA sc id ia d is pa r f rom Sep ha de x LH -2 0 c hr om at og ra ph y
at 266 nm (E dots) and 326 nm (F O O O ), and elution profiles of iron (x x x), titanium (dash line), and
v an ad ium ( so li d l in e) . 1 0m l c on cent ra te d twofol d b y f re ez e- dr yi ng , 1 0m l f ra ct io ns .
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INORGANIC ASPECTS OF THE BLOOD CHEM ISTRY OF ASCIDIANS
16 1
2.0,1.0l
210.5i
450 550
WAVELENOTH IN HITl
F ig ur e 5 a. Ab so rp ti on s pe ct ra o f f ra cti on s 5 Auv s ol id l in e, AV IS
dot s) a nd 11 (Buv d as h l in e, BVIS i rc le s) f rom Sephadex G-75 chr oma -
t og ra phy o f b lo od p la sma o fPyur a c hi le ns is .
U ltrav iolet spectra effra ctions 4 , 7 and 11 from S eph
adex G -75 chrom atography of A . dispar blood plasm a
are shown in Figure 5b. Fraction 4 had an absorption
shoulder at 265-285 nm and also correspon ds to the first
titan ium -band in the respective p rofile (F ig. 2b). F rac
tion 7 show s absorption bands at 210, 260-280, and 326
nm , and it corresponds to the first vanadium band (Fig.
2 b). F ra ctio n 1 1 ( ye llowi sh ) h ad two abs orp tio n maxima ,
at 266 an d 326 nm , respectively, and correspond s to the
th ird iro n b an d (F ig . 2 b).
U ltra vio le t sp ec tra o f fra ctio ns 5 , 8 , 1 7,2 1,2 5,2 9, a nd
36 from Sephadex LH -20 chrom atography of A . dispar
blood plasm a are show n in F igure 6a. Fractions 3-5 had
absorption m axim a at 266-270 and 322-324 nm , corre
sp onding m oreover to the bo rder-line z one betw een the
first iron band and the respective iron shoulder, and to
the first titanium band (Fig. 4). Fraction 8 also had tw o
absorptio n band s, at 262 and 320 nm , w hich only app ear
to be associated w ith the third vanadium peak (Fig. 4).
Fraction 17 had ultraviolet bands at 232 nm and in the
zone of 280 nm , corresponding to the fourth iron peak
in Figure 4. Fraction 21 had one absorption band at 280
nm and two small shoulders at 274-276 nm and 286-
2 88 nm , respec tively. T his fraction also corresponds to
th e first tita nium b an d (F ig . 4 ). F ra ctio n 2 5 h ad u ltra vio
let bands at 230 and 270 nm, and one shoulder at 292-
294 nm . This fraction appears not to be associated w ith
20 0
240 280
32O
360
F ig ure 5 b. U ltra vio let sp ec tr a o f fr ac tio ns 4 C c ir cles , 7 D , E X
do ts), a nd 1 1 ( E so lid lin e) fro m S ep had ex G -7 5 c hro ma tog ra ph y o f
b lo od p la sma o fAs ci di a d is pa r. D i lu ti on s hown we re a pp li ca bl e.
any m etal. Fraction 29 had the following absorption
m axim a: at 210, 232 (shoulder), 270, and 292-294 nm
(shoulder), and should correspond to the sam e group of
com pounds as fraction 25 (have sim ilar UV spectra).
Fraction 36 had three ultraviolet m axim a, at 218 (not
show n), 296, and 328 nm , and it corresponds to the last
iron band (Fig. 4). Fraction 34 had a spectrum sim ilar
to fraction 36, except for the band at 296 nm, which in
fraction 34 appears as a shoulder in the zone of 280 nm .
Also, fraction 34 correspond to the penultim ate iron
p eak (F ig . 4 ). T he v is ib le s pe ctra e ff ra ctio ns o nl y s howed
a bso rp tio n in cre asin g mon oto nic ally w ith a d ec re asin g
wavelength.
U ltra vio le t sp ec tra o f fra ctio ns 6 ,8 , a nd 1 1 f rom S ep h
adex LH-20 chromatography of P. chilensis blood
plasm a are shown in Figure 6b. Fraction 6 had absorp
tion maxima at 280 nm and in the 310-320 nm zone. It
appeared not to be associated with any principal iron
band (Fig. 3) although it is in the borderline of a m inor
iron pea k (fraction 5). F rac tion 8 -9 also ha d tw o ultravi
olet bands, at 270 and 302-306 nm, but are in the first
principal iron peak zone (Fig. 3). Fraction 11 had an ab
sorption shoulder band at 260-280 nm and another that
tends to disappear at 286-288 nm . This fraction is in the
second principal iron peak zone (Fig. 3). In the 12-24
fra ctio n ra ng e, th e a bso rp tio n sp ec tra sh ow ed n o b an ds.
From fractions 25 to 29, the ultraviolet spectra only
show ed one light band at 266 nm . The visible spectra of
fra ct io ns a ls o con sis te d in abs orp tio ns in cre as in g mono
ton ical ly w i th dec reas ing wave leng th .
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16 2
D. A. ROMAN ET AL.
2 0.5
0.2
2. 0
20 0
240 280
WAVELENGTH IN llfTl
32 0
36 0
Figure 6 a. Ultraviolet spectra o f fractions 5 F , 8 G dash
line), 17 ( H X X X ), 2 1 (I O a O ), 25 ( J solid line), 29 (K dots), and 36
( L c ir cl es ) f rom Sep ha de x LH -2 0 c hrom at og ra ph y o f b lo od p la sm a o f
Ascidia dispar.
Discussion
T he a na ly sis re po rte d h ere s ho uld su pp ort th e c on clu
sion that P . chilensis is an iron and titanium accum ula
tor, and that A . dispar is an iron, titanium , and vana
dium accum ulator. In both species the predom inant
m eta l w as iro n, w hic h in th e c ase o f P . c hile nsis is c on sis
te nt w ith a sc id ia n p hy lo ge ny w ith re sp ec t to v an ad ium-
and iron-c ontaining species (H aw kins et ai, 198 3c). In
the order P leurogona, all of its fam ily species are iron
accum ulators (Swinehart et ai, 1974; Agudelo et ai,
1 98 2). Howev er, A . d is pa r a pp ea rs to b e an iro n-p re dom
in an t s pe cie s, a lth ou gh , it a ls o a ccumu la te s t ita nium and
v an ad ium a t g re ate r le ve ls th an con sid ere d n on -b io lo gi
cal (Saxby, 1969; H aw kins et ai, 1983e) w ith respect to
m etal contents in blood cells. Results from the whole
body (specim ens) are not reliable because w hen the ani
m al is rem oved it im mediately begins to lose blood. In
th e su b-o rd ers A plo uso bra nc hia a nd P hle bo bra nc hia ,
the m ajority conta in vanad ium in their blood (H aw kin s
et ai, 1983 e; M ichibata et ai, 1986). Titanium h as b een
reported in dona intestinalis (N oddack and N oddack,
1939) and Eudi sl oma r it te ri (Levine , 1961 ,1962a ,b ), bu t
acco rding to G oodbo dy ( 1 974), th ere is no concrete evi
dence that titanium would be concentrated in blood
cells. In the present w ork evidence is presented of this
m eta l in th e b lo od c ells o f P . c hile nsis a nd A . d isp ar.
H ow ev er, some o f th ese re su lts c ou ld b e o nly a pp are nt
from the biochem ical point of view , because they m ay
b e in flu en ce d b y th e a sc id ia ns immed ia te e nv iro nm en t
200 240
280 320
WAVELENGTH IN 111«
36 0
F igu re 6 b. U ltra vio le t sp ectra of fra ctio ns 6 M d ots , 8 N d ash
l in e) , a nd 1 1 (O s ol id l in e) f rom Sep ha de x LH -2 0 c hr om at og ra ph y o f
b lo od p la sma o fPyur a c hi le ns is .
e.g ., th e flo atin g m eta llic b arrels o f m arin e p oo ls w here
fixatio n occurs. T iO 2 and F e2O 3 are fre quently used as
pigm ents in m any paints (O rn a, 198 0). B ec ause of their
ability to ac cum ulate m etallic trace elem ents fro m sea-
w ate r, tu nic ate s a lso h av e b ee n su gg este d to se rv e a s m a
rine pollution indicators (Papadopoulou and K anias,
1977). Therefore, the Ti in P. chilensis, and the higher
concentrations of Fe and Ti in A . dispar, may also be
a ss oc ia te d w ith th is a sp ec t, ra th er th an b ein g con sid ere d
e sse ntia l e leme nts su bje cte d to se le ctiv e a cc umula tio n
m echanism s. The accum ulation of uncom mon m etals
b y a sc id ia ns in sig nific an t c on ce ntra tio ns is still a n o pe n
question. F or instanc e, som ething sim ilar to w hat h ap
pens to T i, occurs to N b (R ayner-C anham , 1984).
Iron is the predom inant m etal in P. chilensis cyto
plasm , but in A. dispar 26.9% could be in cell mem
branes. T itanium is alm ost distributed likew ise in both
species' cytoplasm and cell m em branes. Vanadium is
predom inant in A. dispar cytoplasm cells, although
23.6% could be bound to m em brane cells. Therefore,
variable fraction of m etals, which m ay depend on the
spec ies, are associated w ith blood cell m em branes of tu
nicates.
B lo od p la sm a o f b o th sp ec ie s w ere n ea rly n eu tra l, w ith
a low er salinity than the habitat seaw ater and w ith low
c on ce ntra tio ns o f su lp ha te io ns. B esid es, th e C a/Mg c on
ce ntration ratios w ere grea ter (0.33 for P . ch ilensis and
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INORGANIC ASPECTS OF THE BLOOD CHEM ISTRY OF ASCIDIA NS
16 3
0.30 for A . dispar) com pared w ith the seaw ater (0.23).
The N a/K concentration ratios w ere low er (20.1 for P.
ch ilen sis a nd 2 2.2 fo r A . d isp ar) th an in seawa ter (2 7.9 ).
C alcium and m agnesium w ere not detected in the cy-
tosolic solutions, and the N a/K concentration ratios
w ere v ery different (4 .6 fo r P . c hilensis and 346.7 for A .
d is pa r). N ev er th el es s, b oth w er e n ea rly n eu tr al a nd th eir
su lp ha te io n c on te nts w ere low, re ac hin g 1 1.7%a nd 6 .5%
o f t he ir c on te nts in p la sm as o f P . c hile nsis a nd A . d isp ar,
re spectively. T his im plies that the low c oncentratio n of
sulphate in plasm as (in respect to the concentration of
su lphate in seaw ater), is not the result of the accum ula
tio n in to cyto pla sm ic b lo od c ell s olu ti on s. Cons id era bl e
co ntroversy still exists on the intracellu lar pH and con
centration of sulphate in the intact blood cells of tuni-
ca tes (D ingley et al., 198 2; H aw kins et ai, 1983a ; F rank
étal.,1986).
To obtain more knowledge about the behavior of
som e m ajor tunicate b lood com ponents, plasm a-cell in
te ra ctio n w as a bru ptly in du ce d in th e b lo od itse lf (d ue to
lack of A . nigra blood, this experim ent w as carried out
o nly w ith P . c hile ns is b lo od ). B lo od c ells a pp are ntly w er e
n ot ly se d u nd er w ho le b lo od ly sis p ro ce du re s, a cc ord in g
to m ic ro sc op e o bs erv atio ns and to d iffe re nt ia l UV -s pe c-
tra of pla sm a, cy tosolic solution and lysed w hole blood
sam ples. T he results (T able IV ), are consisten t w ith the
fact that the blood ce lls of P . ch ilensis are no t acidic and
it seem s that interactions could occur betw een plasm a
and cellular com pounds, that could account for the de
crease of sulphate, calcium , m agnesium , sodium , and
pota ss ium con centra tio ns in whole ly se d b lo od s olu tio n,
in respect to their concentrations in blood plasm a. P art
of these components could be taken up by some com-
pound(s) of the cellular m em branes. It is also possible
th at su lp ha te , c alc ium, a nd m ag ne sium in p artic ula r, in
te ra ct w ith s om e in tra ce llu la r c ompo un ds, w hic h w ou ld
m ean, for instance, that sulphate is consum ed by intra
cellular com pounds of cytosolic solutions. D ue to the
c omple xome tric titra tio n m eth od b y m ea ns o f w h ic h c al
cium and m agnesium were determ ined (Rom an et al.,
1 98 2), it is fe as ib le t ha t in tra ce llu la r s tro ng me ta l lig an ds
take up part of the calcium and magnesium of the
p la sm a. T he re fo re , th is c ou ld b e th e first e vid en ce o f su l
phate consum ption by blood cell com ponents of tuni-
cates, as hypothesized by Hawkins et al. (1983b). It
should explain its low con centration in ascidia n b lood
plasm a a s c om pared to the blood plasm a of other m arine
anima ls (Bu rto n, 1 97 3).
B oth p lasm as an d cytosolic solutions w ere reducing
w ith respect to perm anganate and dichrom ate, respec
tiv ely . H ow ev er, d ep ro te in iz atio n p rio r to th e titra tio n
w ere n ot m ad e. H ow ev er, in th e c as e o f t he b ac k titra tio n
o f d ic roma te m eth od , th e samp le w as a cid ifie d w ith c on
centrated perchloric acid, a d eproteiniz ant (C arr et ai,
1983). In th e pionee ring studie s of E ndean (1985a) sim
ila r a ssa ys w ere te ste d, a nd Muz za re lli (1 97 3) u se d b ac k
titra tio n o f d ic romate fo rc hitin d ete rm in atio n. H awkin s
et al. (1980a) have detected N -acetylam in osugar com
pound s in th e blood pla sm a of tunicates. O ther reducin g
com ponents that have been reported in ascidian blood
include som e reduced form of m etals, the tunichrom e
lik e c ompo un ds a nd th e so c alle d a po fe rre ac id s (M ac ara
et al., 1979a, b, e; Agudelo et ai, 1982, 1983b, 1985;
Hawkins et ai, 1983b; Bruening et ai, 1985; Frank et
ai, 198 6).
M aintaining iron and vanadium in reduced form s in
sp ec ia liz ed b lo od c ells, a nd a lso in some e xte nsio n in th e
plasma in the case of iron (Agudelo et ai, 1983b; Ro
man , u np ub . re su lts from P . p ra ep utia lis), re qu ire d more
in ve stig atio n in a de qu ate ly c on tro lle d a rtific ia l c on di
tions.
T he plasm a spectra (F ig. 1) are sim ila r for P . chilen sis
and A . dispar. The m ain differences are the presence of
a shoulder at 375-385 nm, and the existence of pink-
o range com pound(s) hav ing an absorptio n band at 450 -
475 nm in the plasm a spectrum of P . chilensis. P . stoloni
fera pink compound(s) had a visible band at 497 nm
(H awkins et ai, 1980a). The plasm as UV- spectra of A .
nig ra (K ustin et ai, 1976), A . ceratodes (H aw kins et ai,
1980a ), P od oc la ve lla mo lu cc en sis , P oly ca rp a p ed un cu -
la ta (H aw kin s et a i, 1 98 0b ), a nd P . s to lo nifera (H aw kin s
et ai, 1980a) also have bands at 260-275 nm and 300-
330 nm ranges. A band at 335 nm (Agudelo et ai, 1982)
w as on ly dete cted in p lasm a of B . ovifera. T he m ain sim i
larity o f th e visible spectra of P . chilensis and A . dispar
plasm a is the band at the 675 nm zone.
A nià ³n e xc lu sio n, c atio n re ta rd atio n, a nd o th er p ro b
lems o cc ur in th e c hromo to gra ph y o f m eta l-c on ta in in g
substances on Sephadex G and LH types. This is due to
the sm all am ounts of donor groups present in the m ate
rial (P harm acia F ine C hem icals, 1977; K ura et al., 1977 ;
Johnson and Evans, 1980; LÃ ¶nnerdall and H offm an,
1981). T o m inim ize this prob lem , 0.0 1 M N aC l and 0 .06
M acetic a cid solutio ns w ere used as eluen ts w ith Sepha
dex G -75, and m eth anol/w ate r grad ient w ith S epha dex
LH-20 chrom atography, respectively. Som e level of
m ethanol w as alw ays m aintained in the separative pro
cess and prior to the sam ple run, the colum n w as condi
tion ed w ith m ethanol p.a. A s S ephadex L H-20 w as used
w ith a m ixture of polar solvents, adsorption and parti
tion effects m ust be considered to play m ajor role in the
s epa ra ti on . Ge l f il tr at ion e ff ec ts can be d is rega rded.
T he elution behav ior of plasm as of P . chilensis and A .
dispar from Sephadex G -75, were sim ilar in respect to
absorbance versus fraction collected (F ig . 2b), but the
p atte rn s fo r me ta l c on te nts v ers us fra ct io n colle cte d (F ig .
2b), w ere not sim ilar in function to the sam e m etal con
sidered. In P. chilensis plasm a, evidence of iron com -
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16 4 D. A. ROMAN ET AL.
pound s w it h a h ig h mo le cu la r w eig ht w as fo un d (fra cti on
5-6), in addition to iron bands c orresponding to low m o
le cular w eig ht iron com pounds. H ow ev er, these m ight
c orre sp on d to iro n c ompo un ds o f h ig h mole cu la r w eig ht
that show ed a greater affinity for the gel phase than for
the aqueous p hase. In A . dispar, no evidence of high m o
lecular w eight iron com pounds w as found. H ow ever,
th ese w ere fo un d in th e c ase o f t ita nium (fra ctio n 4 ). L ow
m olecular w eight com pounds of iron and titanium , or
m eta l c ompo un ds th at sh ow ed g re ate r a ffin ity fo r th e s ta
tionary p hase w ere also detected. In both p lasm as vana
dium appears to exist as low molecular weight com
pounds, unless the high m ole cular w eight com poun ds
were re ta rd ed b y ads orp ti on p henomen a.
T he abs orp tio n s pe ct ra o f th e fra ctio ns a ss oc ia te d w ith
h ig h mo le cu la r w eig ht iro n compound s (F ig . 5 a, fra ctio n
5 ), c an no t c orre sp on d to a n F e (III) h yd ro ly tic p olyme r,
w hich only show ed a shoulder at 470 nm (Flynn, 1984).
T he a bso rp tio n sp ec trum o f fra ctio n 1 1 (F ig . 5 a) a pp ea rs
to c orre sp on d to G -7 5 low mole cu la r w eig ht o rg an ic p ig
m en t th at c ou ld b e a tu nic hrome -lik e c ompo un d(s). T he
abso rption spectra of fractions 5 and 11 acco unt fo r the
spectrum plasm a of P . chilensis, so these results appea r
n ot to b e a rtif ac ts .
T he u ltra vio le t s pe ctra o f th e fra ctio ns a ss oc ia te d w ith
app are ntly h ig h mo le cu la r w eig ht tita nium compound s,
from A . dispar plasm a chrom atography on S ephad ex G -
75 (Fig. 5b, fraction 4), only show s a shoulder at 270-
286 nm . This absorption zone w as also checked for the
in dic atio n o f a h ig h mo le cu la r w e ig ht iro n compound (s ),
b ut n o v isib le b an ds w ere o bse rv ed . T he u ltra vio le t sp ec
tra o f fra ctio ns 7 a nd 1 1 (F ig . 5 b) a pp ea re d to c orre sp on d
to c lo se ly re la te d compound s, a pp are ntly o f low mo le cu
lar w eight, associated w ith vanadium an d iron, resp ec
tively. Their UV spectral features suggest that tuni
chrom e-like com pounds m ay also be involved in these
fra ctio ns (B ru en in g e t a i, 1 98 5). In c ompa riso n w ith th e
a bso rp tio n s pe ctrum o f t he w ho le b lo od p la sm a o f A . d is
par (F ig. 1), in the C hrom atographie fraction s, th e ab
sorption peak a t 67 5 nm zone w as not observed.
The elution behavior of plasm a of P . chilensis and A .
dispar on Sep hadex LH -20 w ith m ethanol/w ater gradi
ent, show ed sim ilar patterns for absorbance, and iron
contents versus fraction collected (F ig. 3, 4 ). F or P . chi
le ns is p la sma, Chromatogr aphie e vid en ce o f ir on -c om
p ou nd s w ere o bta in ed , a nd th e s am e o cc urs fo r iro n, tita
nium , and vanadium com pounds in A. dispar plasm a,
re sp ec tiv ely , wh ic h app ea r n ot to b e in org an ic h yd ro ly tic
p ro du cts o f m eta l io ns.
In A . dispar, the absorption spectra of fractions 3-5
(F ig . 6 a) a pp ea r to b e a sso cia te d w ith iro n a nd tita nium-
com pou nds, but a ccording to the spectra of fractions 4 ,
7, and 11 from Sephadex G-75 (Fig. 5b), the titanium
compound(s) should tend to absorb at 260-290 nm
zon e. Iron, vanadium -com poun ds and tun ichrom e like
su bstances also absorb at 32 0-330 nm . T he ultraviolet
sp ec trum o f fra ctio n 8 (F ig . 6 a), sh ou ld c orre sp on d th en
to vana dium com pound (s). T he ultraviolet sp ectrum of
fraction 17 m ay correspond to iron com pounds of pro-
teinaceous nature, due to the band at 280 nm zone, and
the sam e se em s to oc cur in fraction 2 1 for titanium co m-
p ou nd (s). F ra ctio ns 2 5-2 9 (F ig . 4 ) w e re n ot a sso cia te d to
a ny m eta l io ns, a nd b y th eir sp ec tra a pp ea r to c orre sp on d
to closely related com pounds. Fractions 34-36 are re
la te d to iro n, a nd b y th eir sp ec tra l fe atu re s s ho uld c orre
spond to iron com pound(s) sim ilar to those obtained
from th e intera ction betw een iro n and fractions 8-13 G -
75 chrom atography of A . ceratodes plasm a (H aw kins et
ai, 1980a). Therefore, compounds of fractions 3-5
should be closely related to iron com pound(s) of frac
t ions 34- 36 .
In P . chilensis plasm a chrom atography on S ephadex
L H-2 0 g el, fra ctio n 6 (F ig . 3 ) a pp ea r n ot to b e a sso cia te d
w ith iro n, a nd th eir sp ec trum (F ig . 6 b) c ou ld c orre sp on d
to tunichrom e-like substances sim ilar to spectrum of
fraction 11 from Sephadex G-75 (Fig. 5a). How ever,
fractions 8, 9 (Fig. 3) are related to a main iron peak,
then those should contain iro n com po und(s), w hose a b
sorption peaks show (F ig. 6 b) hip sochrom ic shifts in re
spect to the spectrum of fraction 6. Hiper- and hipo-
chro mic effec ts in the bands can also b e observed. F rac
tion 11 is in the zone of the second iron peak (Fig. 3),
a nd b y th eir u ltra vio le t s pe ctra (F ig . 5 a), may corre sp on d
to iro n c ompo un d(s ) o f p ro te in ac eo us n atu re .
It is lik ely th at b y d ilu tio n th e v is ib le a bso rp tio n m ax
im a w ere n ot o bse rv ed in th e sp ec tra o f fra ctio ns c om in g
from L H-2 0 c hroma to gra ph y o f b lo od p la sm as.
T he c omplic ate d h yd ro ly tic p ro ce sse s o f iro n (F ly nn ,
1 98 4), tita nium (P as ca l, 1 96 3, C ia va tta e t a l., 1 98 5) a nd
vanadium (Kustin and Macara, 1982) in a pH media
c lo se to n eu tra lity , su ch a s th e b lo od p la sm a o f t un ic ate s,
su gg ests th at th ese e leme nts c ou ld b e fo un d a s c oo rd in a
tion com pounds w ith proteic or non proteic organic li-
g an ds . T he lig an ds th at h av e b een a ss oc ia te d w ith me ta ls ,
in tunicate plasm a, are proteins (H aw kins et ai, 1980a;
W ebb and C hrystal, 1981 ; A gudelo et al, 1983b) and N -
acetylam inosugar com pounds (H aw kins et al., 1980a,
b ). H ow ev er, A gu de lo e t a l. (1 98 3b ) c on sid ers th at th ese
last co mpounds could corre spond to tunichrom e-like
substances. It also has been sugge sted th at a-hydrox y-
c arb ox ylic a cid s re sid ue s c ou ld b e in vo lv ed in th e m eta l
c omple xa tio n b y tu nic ate s (R ay ne r-C an ham, 1 98 4).
T he m atte r of protein m eta l-binding, and the study of
lig atin g sy stems fo r m eta ls in th e b lo od p la sm a o f a sc id i-
a ns P . chilensis and A . dispar w as scarcely treated here.
H ow ev er, in fo rm atio n w as o bta in ed a bo ut th e p re se nc e
of Fe, Ti com plexes, and likely vanadium com plexes in
b lo od p la sm a o f s pe cie s u nd er stu dy . T he re fo re , it is re a-
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INORGANIC ASPECTS OF THE BLOOD CHEM ISTRY OF ASCIDIANS
165
so na ble to su pp ose th at th ey a re in vo lv ed in th e d yn am ic
p ro ce ss es (sto ra ge /c arrie r) o f m eta ls in tu nic ate b lo od .
Accordingly, the high m olecular weight m etal com -
p ound (s ) s ho uld b e tra ns fe rrin -lik e me ta llo pro te in s,
which has been recently show n in the blood plasm a of
P . stolo nifera (M artin et ai, 1 984; F inch and H uebers,
1986).
Betw een pH 2.5-3.5, tunichrom e solutions appear
green, due to the broad band in the zone near 660 nm
(M acara et ai, 1979b). W e found an absorption peak
around 675 nm in both plasmas and in fraction 5 from
S ep ha de x G -7 5 c hroma to gra ph y o f P . c hile nsis p la sm a.
T his sh ou ld a ris e from iro n-c ompo un d(s) o f h ig h mole c
ular w eight w ith respect to th e exclusion lim it of the gel.
It has recently b een suggested that in the d-d transitio n
energy at 660 nm zone, tw o nitrogen atom s from the co
o rd in atio n b y lig an ds lik e D -g lu co samin e (M ic era e t a i,
1985) could be involved. This w as not observed w hen
the amino group was protected, as occurs in the case
o f N -a ce ty l-D -g lu co samin e. T he re fo re , m ore a tte ntio n
should be focus in the tunicates blood com pounds that
sh ow a bsorption bands at 6 60-675 nm zon e, due to their
p ote ntia l a ss oc ia tio n w ith me ta l b in din g.
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