Coenzyme Q homologs and trace elements content of Antarctic fishes Chionodraco hamatus and Pagothenia bernacchii compared with the mediterranean fish Mugil cephalus

Camp. Bi~>chem. I’hyskd. Vt~i. 118A. No. 4. pp. 977-980. 1997 Ctjpyright 0 1YY7 Elsev~er Science Inc. All rights reserved.

MN 0 100.C)6?9/97/$1 7.00 rll S03@0-Y629(97)0c70c74-?

ELSEVIER

Coenzyme Q Homologs and Trace Elements Content of Antarctic Fishes Chionodraco hamatus and Pugothenia bernucchii Compared With the Mediterranean Fish Mugil ceghalus B . Giardina~

M. L. Gozzo,JF B. Zappcosta,” L. Colacicco ,* C. CalliLT A. Mordente, f and S . Lippa”

*INSTITUTE OF CHEMISTRY AND CLINICAL CHEMISTRY, ANL) ~INSTITUTE OF BIOLWIC.AL CHNISTW, FACULTY OF MEDICINE, CATHOLIC UNIVERSITY OF ROME, ITAL.)

ABSTRACT.

&enzyme

&a~-o humatus)

were

p,lrticular.

muscle

determined.

Q and trace element

investigated

and erythrncyte

Antarctic with

M. ce@~&s.

temperate

fishes,

d&rrnce

in the crystallization

only

zinc and selenium,

protein

of a srlrni~lm-c~)ntaining

antarctic

existing

that,

isolated

iI high level of antioxidant

species,

ered as a stimulating

basis for further

PHYSIOL 118A;4:977-980,

1997.

0

metaholism,

Pqothmic~

cephalus,

brnlacchii,

Mqil

Science

cnensyme

phybico-chemical

characteristics

of Antarctica

make

Thus,

the extrrme

some

evolutionary

biochemical

conditions

of this habitat

adaptations,

levels

that,

hoth

very

often,

properties

of antarctic

temperature constantly

td the oxygen-rich coastal at - 1.87”C, the equilibrium

water

and

mechanisms tifreeze”

ice.

Hence,

of some htmatological erythrocytes

and

in peculiar the

Antarctic ocean is temperature of sea

fishes

have

lower the freezing

developed of “antempera-

(decreased

concentration)

number

to compensate

of

acid sequence the results

tL) the meditrrranean

in

to the Icvcls

of

in the liver

closely

aunila1

indicate,

tar the

hhh and may hc conyid-

of Antarctic

organisms.

(X)CII’ HIO(‘HEAI

E, \elcnium.

zmc, copper.

(:hir~nr&tcr~

hLltnccnc.\,

specie\

As this latter

of blood

aspect

cytes and hence

viscosity

is concerned,

of hemoglobin

stage of such evolutionary ant of warmer

to their

temperatures

ture of only a few degrees effects (7). A well-known pointed

utilization

(5,16).

aspect

This

tissues

trace elements

environment generally of

of

caused trom

the

lipid

different

in temperaleads tc> lethal

antarctic

t&es

metah
us to investigate

that are known

antarctic

and intolt‘r-

so that an increase

to the oxidative

preferential

(7).

the extreme

On the whole,

centigrade

characteristic

our attention

he c~msidered

strategy.

fishes are fmely adjusted

at low temperatures

the total lack of crythmin the hl,)od of tishes of the

could

Channichthyidae

antarctic

that is their

pathway

the presence, tishes,

to he essential

of those

for the func-

tion of antioxidant enzymes (2). Along the SWNA line, particular attention has been given to the presence of cot’~l:yme Q and vitamin E. In fact, it is well known that the oxidative metabolism of the cell may lead to the production of some’ reactive

1996.

the high

on the prc.rcnce,

hy an amino

in various

way, and the modification

characteristics

and hemoglobin

resulted

such as the synthesis

which

in a noncolligative

at physiological

Ohserved related

Inc.

family

ahout

As to the fish fauna,

antarctic

of cold adaptation

(glyco)peptides,

ture of hlnod

organisms.

brought

Moreover,

report

for the increase

this environment ideal for studying temperature adaptations at the various levels of organization of a given organism.

h,lb heen

All together,

metahollbm

Q, vitamin

oxygen

finding

In

zinc were

zinc and copper

Q form generally

and G)Q,,.

heart.

with respect

INTRODUCTION

The

ovine

on the oxidative

reactive

(:oQIo

and Chiono-

cephulus).

cc~pper and

levels of selenium,

particular

IS characterized

from

1997 Elsevier

KEY WORDS. (&dative

between

defenses

studies

higher

This

fish (Mugil

E, selenium,

very well with a recent

in additic)n,

protein

Q, vitamin

marine

ot Q ic, the coenzyme

QLI wah found.

correlate

temperate

muscles,

in the place

coenzyme

temperature

ci one

of coenzyme

fishes,

in C. hamatus,

of thib fish, of a rinc-hinding

that

at the level of their

homolog

ohserved

with

concentrations

In antarctic

the

of two antarctic tishrs (Pagotheniu bernucchii

contents

compared

fishes showed,

c,mpared

to that

and

may attack membranes

oxygen

species

that directly

sensitive macromolecules and chr~matin.

that

or indirectly comprise

ccl1

R. Giardina et al.

978

Because

oxygen-derived

chemical

species

that

free radicals

may have

the biological

structures

in the

of evolution,

course

many defense

are highly

several

reactive

harmful

effects

of the cell, it is not surprising all organisms

mechanisms

that involve

have

on that

analyzed

developed

the participation

of

different enzymes and cofactors acting as radical scavengers. The main protective enzyme systems that either bypass free radical

generation

chain

or interrupt

peroxide

dismutase,

addition,

the key role of coenzyme

respiratory

chain

combination

with

vitamin

E (13).

of these

to the presence

tissues from two species nucchii and Chionodraco

aspects,

of copper,

structure in

evolu-

we give spe-

zinc and selenium

of coenzyme

Q in various

of antarctic fishes (Pugothmia hamatus) in comparison with

fish (Mugil cefihalus).

zyme Q is also related

In

antioxidant

For the obvious

particular

and to the in viva concentration

mediterranean

and catalase.

to its quinon-like

The

to the evidence

interest

that

berone

in coen-

its in &o

con-

centration appears under the control of oxygen tension and seems to be involved in cold-exposure adaptation mechanisms. The results

compared

the temperate

fish, M. cephalus, seem to possess some inter-

esting

evolutionary

with those

obtained

on the red blood

with 0.9% NaCl solution.

in parallel

on

implications.

AND METHODS tishes (I’. bemchii

Two antarctic mediterranean

tish

study.

All specimens

About

1 g of muscle

(M.

tamin

were stored

at -80°C

and Kunkel-Stauffen).

were used to determine

until

Aliquots

coenzyme

then panol

precipitated

(95 : 5) and coenzyme

hexan.

The

a stream

extract

E, selenium,

of nitrogen,

chromatographed

evaporated

redissolved

on an HPLC

was digested (MLS- 1200

in acidic medium Mega Milestone

Table

1 reports

nium,

zinc and copper

examined.

cited.

main

feature

form of coenzyme

fish tissues.

Thus,

of the coenzyme,

teristic

is the complete

that of P. bernmchii

is the

M. cephulus, as other

mediterra-

by the presence

in the muscle

of antarctic Between

ofC.

Q content

apparent

antarctic

form (2,) was found.

the coenzyme

E, sele-

of all species

characterizes

nean fishes, is characterized homolog

immediately Q that

whereas

(2, vitamin tissues

of the Qlc form fishes, only the

the antarctic

tishes,

humutus, whose main charac-

lack of hemoglobin,

is about

2-fold

and M. cephulus.

Of the trace elements examined, the most interesting seem to he selenium and zinc, which show muscle levels four to five times higher

in the case of antarctic

fishes with

respect to the temperate fish M. cephnlus. A significant increase, although not so marked, was also seen in the copper which

in both antarctic

that observed

in M. cephalus

Muscle

levels

species

is about two times

tissues.

three

times

on red blood

The

same

cells only

for M. cephalus a concentra-

parameters

were

mea-

in the case of P. bemucchii

(Table 2). High levels of zinc and coenzyme Q were found, whereas vitamin E is below the detection limit of the instrument.

E, selenium,

with

DISCUSSION The relative

weight

of the two main metabolic

: isopro-

muscle cells, anaerobic

5 ml of

tory chain, conditions

to dryness (Beckmann)

under and ac-

with a microwave syss.r.l., Bergamo, Italy). stripA/S,

as described by Gozzo et al. ( 10,111. with the same apparatus but in con-

stant

analysis

as described

The

different

in the muscle

sured

Copenhagen, Denmark) Selenium was measured stripping

the levels of coenzyme

analysis.

homogenizer

Zinc and copper were determined with computerized ping potentiometry using the Tracelab (Radiometer

current

Q, vi-

RESULTS

higher.

cording to Lippa et al. (12). Vitamin E was measured with the method of Ericson and Soerensen (9). For trace element determination, an aliquot of homogenate tem

coenzyme

with the methods

tion

in 100 ,~l of ethanol apparatus

zinc and copper

for this

of ethanol

Q was extracted

was then

in 2 ml of the same

were used to determine

E were similar

Q, the homogenate (about 100 with 0.25 M SDS. Proteins were

with 2 ml of a mixture

were twice

C. humutus showed

zinc and copper. To determine coenzyme mg of proteins) was treated

parameters

whereas

of the homogenate

Q, vitamin

resuspended

of Lowry

were washed

of vitamin

was homogenized

with an ultraturrax

Cells

and P. bernucchii,

selected

tissue from all species

in 2 ml of 0.25 M sucrose (Janke

were

the same

and one

and C. hamatus)

cephalus)

by the method

cells.

and then

Aliquots

content,

MATERIALS

were determined

are su-

Q in the mitochondrial

it to work as a liposoluble

importance

cial attention

peroxidase

(4) is linked

that also enables tionary

glutathione

the chain

al. (3). Proteins

et al. (14). In the case of P. hernacchii,

by Chi Hua et

volved

glicolysis

is conditioned by environmental that may modulate the tissue

enzymes,

substrates

pathways

and mitochondrial

and cofactors.

of

respira-

and individual levels of the inThe preferential

utilization of the lipid metabolic pathway observed in antarctic fishes (5,6,8,16) suggests a major activation of the oxidative metabolism that, in turn, may lead to a significant production of lipoperoxides and oxygen free radicals. This hypothesis seems to be supported by an overall increase of the antioxidant defenses that characterize both antarctic t;shes as outlined by the level of selenium, a cofactor of glutathione peroxidase and that of zinc, a cofactor of superc’xide dismutabe.

In the respect,

it is suggestive

that, among the

f;shes examined, M. cephaku seems to have a lower degree of antioxidant prc)tection; however, for an overall balance of

Q in Antarctic

Coenzyme

TABLE 1. Coenzyme

Fishes

979

Q homologs

and trace elements content of muscle tissues of Antarctic Mud

Cd&

(ng/mg

defense,

tishes

seem

32.2 2 6.3

27.5 2 6.1 27.6 2 5.4

X6.0 + 9.1

3.0 i- 0.5 9.0 2 3.7

5.2 t- 1.4 35.0 2 9.3

5.9 + 2.4 48.0 ? 9.7

2.2 t 0.6

8.5 -c 4.3

6.6 + 2.4

from those

tectahle.

protein

feature

of aspartate

ported groups

zinc

more

weakly

thionein,

nium

than

the physiological

are paralleled

significance,

to that of a selenium-containing

the amino

in some respects,

tamin

source

these

of coenzyme increase

two molecules

the reduced

is still

is the oh-

of this protein isolated

E. This is prohahly levels

the reason of coenzyme

Q is normally

of vitamin

E. Thus,

are mutually

depen-

form of coenzyme

of :llfatocnpherylquinone,

Q is the main

production

it should

of these

Another

data

species.

a significant

the site of utilization

hy

explanaticm of this finding could reside in some differences in the physical properties of the two homc~logs. In fact, WC‘ may observe

that

tishes,

ture of 9.7”C. tant

coenzyme

meaning

Valur~ XT nwanr i St1 of three determinarions
pre\cnt

hy a crystallization

This physical

hioloqical

constant

with

cc~ld

respect

especially

for homothermic

- 1 .PC, the

the

point

in

Restive

that

in antarctic

whose

temperature

higher

than seawater

Therefore,

tishes,

of crystallization temperature.

Q,,.

crystallization

this could

in the

temperature

is signiticantly

functicming of the respiratory chain for the whole organism. In this respect,

fluidity

phosphorylation,

of coen:yme

Q~~~YJ, where

he excluded,

have an impor-

to a water

temperature

crystallization cannot

organisms. subjected

in temtcmpera-

to membrane

and to the <)vcrall rate cjf the oxidativc

hody

ND 0.5 -t 0.2 96.0 2 11.1 5.9 ? 1.7

Q1,, normally

is characterized

than

1.5

tishes that apis found in A possible

of

Pagothenia bemacchii

of antarctic

cells of the

Q, also possess

a high

feature

Qq form of coenzyme Q rather than QIo, which cells cd temperate fishes and of most vertebrates.

why C. hamatus, which

TABLE 2. Coenzyme Q homologs and trace elements content of red blood cells of Pagothenia bemacchii

Vitamin E (ng/mg prot.) Copper (ng/mg pmt.) Zinc (ng/mg prot.) Selenium (ng/mg pmt.)

oxygen

that

is the presence

fishes,

form of vi-

5.0 i

with that

to the lack of

in their

micrOeilVir(~IIInents

(ng/mg pmt.)

of reactive

be recalled to reach

characteristic

pears from these

compound

C0Q

Qq and vita-

fishes to use the skin as a gas exchange

case of antarctic

the oxidized

comzyme

of C. hamatus compared

surface. of

binding

protein

the ability

perate

in the content

has the highest

re-

metallo-

mobilized

acid composition

by a concomitant

because

of Or is allowed

imply

( 15).

heart

An increase

reductant

amount

in this respect,

is similar

dent

respect,

would

may he

Q.

a specific C& carrier. Hence, 0, supply to tissues is essentially hased on the physical diffusion of this gas, which could reIn this

groups

higher

in

vitamin

protection

in P. hernucchii could well he related

sult in an increased

of a specific

that

in muscles

that,

where

levels of coenzyme

the significantly

with

carboxyl

he recalled

the antioxidant

it has been re-

that the high levels of zinc and sele-

0f particular

accompanied

and a high

role of this protein

servation from o\inc

zinc-binding

this would

amino

hy the presence

Moreover, observed

was not de-

absent,

it should

that of pig tissues,

just by high

which residues

a more rapidly

accomplished min E content

cysteine,

E. However,

E is practically

in the ice fish C.

Because

acidic

a recent

metal more easily than a “normal”

it IS suggestive

protein.

with

level of vitamin

has characteristics

of interacting

carrying

thus providing

zinc. Although unclear,

of cystein

is capable

a zinc-protein

lease the hound

found in tissues from

that

and glutamate.

thilt

both

some cases, as for example

of the C. hamatus

is the low content

content

factors,

very well with

of metallothionein,

A peculiar

protein

selenium

the presence,

of a zinc-binding

distinct

additional

54.4 + 8.3

specimens for each ?I~CCICY

be considered.

to correlate

(15) concerning

nbservaticm hamatus,

should

ofzinc and

The high levels

that

some other

and non-proteic,

antarctic

Chionodraco hamatus

Pagothenia bemacchii

ceDhdus

Valuc~‘,IC ,nc;,rP+ ~ SD of three determinati<>ns on three Jiftrrent

the antioxidant

fishes

36.0 2 8.1

pot.)

Co(zq (ng/mg pot.) Vitamin E (ng/mg pot.) Copper (ng/mg pmt.) Zinc (ng/mg pror.) Selenium (ny/mg pot.)

proteic

and Mediterranean

impair

lower In the of the the right

with grent troubles it is particularly sug-

Q,,, is substituted (0.5%)

hy Q,,

i.s only slightly


the pres-

ence ofQ,, instead of QIO seems to he the result of an adaptative strategy hy which the efticiency of one ci the main metabolic pathway could he maintained at a reasonable level. In addition, living

we are called

in temperate

to consider

environments,

that

m organisms

“c~Jd exposure“

leads to

B. Giardina

an increase

of coenzyme

of an adaptation

Q biosynthesis,

mechanism

probably

(1). However,

as result

this does not

apply to antarctic fishes, as shown by the coenzyme Q levels in their tissues. Indeed, it should be considered that antarctic organisms

live in one of the most stable

and are not exposed coenzyme

to temperature

Q variations

ture changes

than

changes.

environments In other words,

appear to be linked more to tempera-

to cold per se.

The financial support of the Italian National Profframme for Antarctic Research is Fe& acknowkdged.

References 1. Beyer, R.E.; Noble, W.M.; Hirschfelcl, T. Coenzyme Q (Ubiquinone) levels of tissues of rats during acclimation to cold. Can. J. Biochem. Physiol. 40:511-518;1962. 2. Cassini, A.; Favero, M.; Albergoni, V. Comparative studies of antioxidant enzymes in red-blooded and white-blooded antarctic teleost fishes Pugothenia bernacchii and Chionodmco hamatus. Corny. Biochem. Physiol. 106C:333-336;1993. 3. Chi Hua, C.; Jagner, D.; Renman, L. Determination of selenium by means of computerized flow constant current stripping at carbon fibre electrodes. Anal. Chim. Acta 197:25764;1987. F.I. Development of concepts for the role of ubiqui4. Crane, nones in biological membranes. In: Lenaz, G.; Barnahei, 0.; Rahhi, A.; Battino, M. (eds). Highlights in Ubiquinone Research. New York: Taylor and Francis; 1990:5-17. E.L.; Sidell, B.D. Polyunsaturated fatty acids are me5. Crockett, taholized by both mitochondrial and peroxisomal pathways of antarctic fishes. Antarctic J. 27:143-145;1992. E.L.; Sidell, B.D. Substrate selectivities differ for he6. Crockett, patic mitochondrial and peruxisomal beta-oxidation in an antarctic fish, Notothenia gihherifrons. Biochem. J. 289:427433;1993. M.; Giardina, B. Oxy7. di Prtsco, G.; Condo, S.G.; Tamhurrini,

et al.

gen transport in extreme environments. Trends Biochem. Sci. 16:471-474;1991. in a swim8. Eastman, J.T.; De Vries, A.L. Buoyancy adaptations bladderless Antarctic fish. J. Morphol. 167:91-102;1981. B. High performance liquid chroma9. Ericson, T.; Soerensen, tography of vitamin E. Acta Pharmacol. Suet. 14:478-48.3; 1977. G.; Morosi, R. Computer10. Gozzo, M.L.; Lippa, S.; Barbaresi, ized stripping potentiometry applied to copper determination in plasma and urine samples. Trace Elements and Free Radicals in Oxidative Diseases. Chamonix, April 5th-9th, 1993. L.; Call& C.; Barbaresi, G.; 11. Gozzo, M.L.; Lippa, S.; Colacicco, Giardina, B. Computerized stripping potentiometry applied to zinc determination in plasma and urine samples. Fourth Lnternational Congress of International Society for Trace Elements Research in Humans. Taormina, Sept. 25-28, 1995. G.P.; Oradei, A. Determinazione routin12. Lippa, S.; Littarru, aria de1 coenzima Q,,, mediante HPLC in campioni biologici. 1,’ Conferenza Nazionale su La cromatogratia liquida ad alta risoluzione in analitica clinica :situazionc attuale e prospettive. Verona, 1985:51. G.P.; Pe Sole, I’.; Lippa, S.; Oradei, A. Study of 13. Littarru, quenching cd singlet oxygen by coenzyme QId in a system of human leukocytes. In: Folkers, K.; Yamamura, Y. (eds). Biomedical and Clinical Aspects of Coenzyme Q, Vol. 4. New York: Elsevier Science Publishers; 1984:201-208. N.J.; Farr, L.; Randall, R.J. Pro14 Lowry, O.M.; Rose&rough, tein measurement with the Folin phenol reagent. J. Riol. Chem. 193:265-275;1951. 15 Scudiero, R.; De Prisco, P.P.; Camardella, L.; D’Avino, R.; di Prisco, G.; Parisi, E. Apparent deficiency of metallothionein in the liver of the antarctic icefish Chionodrmo hamatus. ldentitication and isolation of a zinc-containing protein mllike metallothionein. Ct~mp. Biochem. Physiol. 103B:201-207; 1992. E.L.; Driedzic, W.R. Metabolic charac16 Sidell, B.L).; Crockett, teristics of muscle tissues from antarctic tishes. Antarctic J. 23:138-140;1988.