pyura chilensis - heavy metals

8/19/2019 Pyura chilensis - Heavy Metals

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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 BÃ¡s 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 sâ€”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 -5Â°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 10Â°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 -5Â°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


5/13

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

â€¢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.7.014.6â€”Cytosolic

o lu ti on o f

A. ispar*n.m.n.m.n.m.0.051n.m.n.m.0.1040.300.027.36346.7â€”

* 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 AUVâ€ ¢€¢€¢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 .


6/13

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


7/13

16 0

D. A. ROMAN ET AL.

22.^3.8

l Â°Â°Â°°,.

0 00

0000

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oo0/VÂ°oo0Â°0OoÂ°

Â°Â°Â°,

o

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oo

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o o

O o

o o

O o

o o

O o

o

o

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o

o

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=00Â°

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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 .


8/13

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 .


9/13

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 111Â«

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


10/13

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

Ã©tal.,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 niÃ 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 -


11/13

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.

Lit era ture C it ed

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