HPLC Analysis of Retinoids Extracted from the Planarian, Dugesia japonica(Physology)

6 Pages·1992·2.1 MB·English

by  Katsu Azuma

ZOOLOGICAL SCIENCE 9: 941-946 (1992) © 1992 Zoological SocietyofJapan
HPLC Analysis of Retinoids Extracted from the
Planarian, Dugesiajaponica
Katsu Azuma1 Naohiko Iwasaki1 Masami Azuma2
, , ,
Takao Shinozawa3 and Tatsuo Suzuki4
1Department ofBiology, Osaka Medical College, Takatsuki, Osaka 569,
2Department ofHealth Science, Osaka Kyoiku University, Osaka 547,
3'Department ofBiologyical and Chemical Engineering,Faculty of
Engineering, University ofGunma, Kiryu, Gunma 376, and
4Department ofPharmacology., Hyogo College of
Medicine, Nishinomiya, Hyogo 663, Japan
—
ABSTRACT Retinoids extracted from the planarian, Dugesia japonica were analyzed by high-
pressure liquid chromatography (HPLC). Al\-trans retinal, all-/ram- retinol, and a\\-trans retinyl ester
weredetectedintheextractsfromtheheadandtailpiecesoftheworm,while ll-cisretinalwasdetected
in theextractsfrom the headpieces. The amountsofa\\-transretinal, ll-c«retinalandall-fransretinol
includingthe retinylesterwere0.1-1.1, 0.11-0.19, and 20-50pmol/head, respectively. The planarian
containedmanyoil-dropletswhichemitted thegreen-yellowfluorescenceprobablyderivedfrom retinol
andretinylester. These resultssuggest thattheplanariancontainsal\-transretinolandtheretinylester
in oil-droplets and ll-cis retinal as the chromophore ofthe visual pigment in the eye.
INTRODUCTION chemical experiments, it has been reported that
the photopigment ofthe planaria {Dugesiajaponi-
The planarian is one of the lowest metazoans. ca) is a chromoprotein which possesses retinal-
The eye of the planarian consists of pigmented dehyde as the chromophore [4]. Recently an
cells and photoreceptors of a microvillar type [1]. immunochemical study suggested the presence of
Extracellular microelectrode recordings from the rhodopsin-like protein in the headofthe planarian
eye of the planarian Dugesia tigrina suggested the Dugesia japonica by use of anti-frog-rhodopsin
presence of a rhodopsin-like photopigment whose rabbit IgG [5].
absorption maximum was at about 508nm [2]. Although ll-cis-retinal isthe most ubiquitous as
Spectral phototaxis experiments showed the sensi- the chromophore in the vertebrate and inverte-
tivity maximum of the planarian eye (Planaria brate rhodopsin [6], a variation in the chromo-
lugubris) at about 475 nm [3] and 530nm for phore of visual pigment is found in other spe-
Dendrocoelum lacteum [3]. The differences in cies: ll-cis3-dehydroretinal isfound in manyfresh
those maxima may have been caused by a con- water vertebrates [7, 8] and invertebrates [9, 10];
tribution from the dermal photoreceptors [2], an ll-cis 3-hydroxyretinal is found in the insects [11];
effectofscreeningpigments [2] or,perhaps,simply and ll-cis 4-hydroxyretinal is in a bioluminescent
a species difference. At present, the visual pig- squid [9]. In addition aM-trans retinal and 13-a's
mentoftheplanarian has hardly been investigated retinalareseeninHalobacteriumhalobium [12]. It
by spectrophotometric methods, because the isunknownwhetherornotthechromophoreofthe
worm contains so little visual pigment. In histo- visual pigment of the planarian is ll-cis retinal.
The purpose of this study is to estimate the con-
Accepted June 8, 1992 figuration of the chromophore of the planarian
Received April 6, 1992 visual pigment by high-pressure liquid chroma-
942 K. Azuma, N. Iwasaki et al.
tography (HPLC) analysis of retinoids extracted rate hexane solvent and served for the extraction
from the worms. Our results indicate that 11-cis of retinoids by the oxime method as described
retinal is one of the most plausible candidates for above. The obtained solution (dichloromethane/
the chromophore. hexane extract) contained retinaloxime and retinyl
ester. After evaporating solvents of the extracts
MATERIALS AND METHODS mentioned above, the residues were dissolved in
50fA of hexane/diethylether/ethanol (90/10/0.1,
vol/vol) and analyzed by HPLC. All procedures
Materials were carried out under dim red light.
The planarian worms, Dugesia japonica were
collectedfromstreamsinthesuburbsofKyotocity Detection ofretinoids in the samples
(Kyoto prefecture, Japan) and Kiryu city (Gunma Extracts ofretinoids from the planarian samples
prefecture,Japan). Kyotowormsweremaintained were analyzed by the HPLC method as reported
by feeding on fresh beef livers, and used for previously [15]. An HPLCsystemequippedwith a
extractions ofretinoids from theirwhole bodies or 4.6x250mm column of YMC-Pack A-003-3 SIL
both the head pieces (anterior part containing the (Yamamura Chemical Labo. Co. Ltd., Japan) and
eyes) and the tail pieces (the tissues without the a pump (TRI ROTER, JASCO, Japan) was used.
head). Head pieces ofkiryu wormswere stored as Theeluentwasamixtureofn-hexane, diethylether
frozen materials and used for extraction of re- and ethanol (90:10:0.1, vol/vol) and was used at
tinoids. the flow rate of 1.3 ml/min for 50min. The
absorbances ofthe fractions at 350nm and at 280-
Extractions ofretinoids 500nm were measured with a detector UVDEC-
Usually, retinoidswere extracted from the fresh 100-III (JASCO, Japan), and with a multi-
or frozen head pieces by the oxime method which wavelength detector MULTI-340 (JASCO,
was developed to extract the retinal from biologic- Japan), respectively. The measurement with
al materials as retinaloximes (syn- and anti-forms) MULTI-340was carried out in order to obtain the
in the original isomericconfiguration without ther- absorption spectra of the fractions over the wide
mal isomerization [13, 14]. The planarian samples range of wavelengths, although the sensitivity of
were homogenized in a solution of 100mM the detector was less than that of the UVIDEC-
NH2OH (pH7.2) and methanol (final concentra- 100-III detector.
tion ofmethanolwas60-70%) usinga homogeniz- Quantities of several retinoids were estimated
er (Physcotron NS-50, Nichion Irikakikai Seisa- from their abosrption coefficients and the peak
kusho Co. Ltd., Japan). The homogenate was areasofknown amount ofstandard retinoids. The
mixed with dichloromethane and n-hexane (1:2, fractions of 2-8min (retention time) under our
vol/vol), shaken vigorously and centrifuged at HPLC conditions were used as the sample of
2,500r.p.m. for 15 min. The upper layer retinylester. The solvent was evaporated from the
(dichloromethane/hexane layer) was collected. fractions and the residue was incubated in 6%
This extraction was repeated three times. The KOH-methanol solution at 25°C for 1 hr for sapo-
collected solution was stored as extracts of re- nification. The amount of retinyl ester was calcu-
tinoids. lated from that of retinol produced by the sapo-
In a few cases, the planarian samples were nification.
freeze-dried for hexane extraction of retinoids.
The freeze-dried samples were shaken vigorously Observation offluorescentimages ofoil-droplets in
planarian tissues
in hexane solvent and centrifuged at 2,500r.p.m
for 15 min. The supernatant was collected and The planarian worm of 5 mm in length was put
hexane extraction was repeated 3 times. The on non-fluorescent slide glass, covered with a thin
collected solution contained retinol and retinyl coverslip and spread by the squash method. The
ester. Precipitates were gently aspirated to evapo- fluorescent images ofthe oil-droplets in the spread
HPLC Analysis of Planarian Retinoids 943
340 380
10 min Wavelength (nm)
Fig. 1. (a) HPLCprofile ofretinoidsextracted from 18bodiesofplanarianworms, (b) Absorption spectraofpeak
fractions indicated by numbers 1 and 2. The worms were dark-adapted overnight in aged tap water at 20°C.
Extractions were carried out by the oxime methods.
specimen were observed using a flouorescence
microscope (Olympus inverted-microscope, IMT-
2, equipped with Olympus incident-illumination
type fluorescence apparatus, IMT2-RFL). The
W
specimenwasexcitedbylight(50 halogenlamp)
passing through an excitation-filter (UG-1) and
observed through a filter (L420) and a dichroic
mirror (DM 400). The photograph of fluorescent
images was taken using the colorfilm (Fujichrome
DX400D).
RESULTS
Retinoids detected in theplanarian
Figure la shows an HPLC profile of the extract
ofretinoids from the whole bodies of 18 planarian
worms of about 10mm in length. This figure was
obtained by recording the absorbaces of the frac- Fig. 2. Fluorescent micrograph of oil-droplets in the
tions at 350nm with the UVIDEC-100-III detec- planarian body. The specimenwasobtained by the
tor. Numbers 1 and 2 indicate peaks close to the squash methods. Bar=50,um.
retention time of standard syn all-trans retinalox-
ime and all-rrans-retinol, respectively. Two big nm), respectively. The fractions between 2 and 8
peaks between 1 and 2 are not identified. The min, which contain retinyl esters, were collected
relative absorption spectra (300-420nm) of the and saponified as described in Materials and
fractions corresponding to peaks 1 and 2 were Methods. Then the material obtained after the
obtainedbythe MULTI-340detector, indicated as saponificationwasanalyzedbyHPLC. Fromthese
curves 1 and 2 in Fig. lb, respectively. The analyses, the amounts of all-trans retinal, all-trans
absorption maximaofcurves 1 and2clearlymatch retinolandall-transretinylesterwerecalculatedas
those of the standard syn all-trans retinaloxime 2.5, 14.7 and 99.4pmol/body, respectively.
(Amax=358nm) and all-trans retinol (Amax=325 Figure2 shows fluorescent images of several
944 K. Azuma, N. Iwasaki et al.
oil-dropletsintheplanarianspecimensobtainedby
the suqash method. The light color offluoresence
was green-yellow suggesting the presence of re- A350 3
tinol and/or retinyl ester. Probably, the planarian |o.002
worms store the dW-trans retinyl ester in oil-
droplets, because the worms contain large
K^j^J[
amounts of the retinyl esters, as mentioned above
'n'V-V.M^yJ^/ ^•V>-A,
(more than 85 mol% of total retinoids). 10 20 30 40 50
Figure3 shows HPLC profiles of extracts from Time (min)
the head (a) and the tail (b) pieces of 28 planar-
ians. Peaks numbered 1 and 2 are corresponding
to syn &\\-trans retinaloxime and al\-trans retinol,
respectively, as estimated from their retention
times and abosorption spectra (data not shown).
Thus &\\-trans retinal and all-trans retinol were
found in the head and tail pieces ofthe planarian.
A350 340 380
Wavelength (nm)
0.004
Fig. 4. (a) HPLC chromatogram ofretinoids extracted
from head pieces of the planarian worms, (b)
Absorption spectra of peak fractions indicated by
numbers 1,2and3. Theplanarianheadpieceswere
separated from 600 bopdies ofthe planarian worms
under room light, then dark-adapted overnight in
aged tap water at 20°C and stored at —20°C until
use. Extractions were carried out by the oxime
methods.
LlaJ UJ
F1i0g.m3i.n HPLC profile of reti1n0oimdisnextracted from both plle-ackiss r1e,t2inaanldox3imaer,escylnoseallt-otrtahnossereotfinsatlaonxdiamredasnydn
the head (a) and tail (b) pieces of 28 planarian a\\-trans retinol, respectively. The relative absorp-
bodies. Thewormswere dark-adapted overnight in tion spectra offractions corresponding to peaks 1,
aged tapwaterat20°C, separated into head and tail 2 and 3 are represented as curves 1, 2 and 3 in
piecesunderdimredlightandwereextractedbythe Figure 4b respectively. Curves 2 and 3 are due to
oxime methods.
the absorption spectraofsyn a\\-transretinaloxime
and a\\-trans retinol, respectively, as indicated in
Retinals in theplanarian headpieces Figure 1. Curve 1 seems to be corresponding to
Figure 4a shows an HPLC profile of the extract the absorptionspectrumofsyn ll-cisretinaloxime,
of retinoids from head pieces. The head pieces because the shape ofcurve 1 is different from that
were cut off from the 600 bodies of the planarian of surve 2 due to syn a\\-trans retinaloxime. The
under room light, then dark-adapted overnight in amounts of all-trans retinal, ll-cis retinal and all-
—
aged tap water at about 20°C and stored at 20°C trans retinol including the retinyl ester in different
until use. The chromatogram was obtained by preparations were 0.1-1.1, 0.11-0.19 and 20-50
recording the absorbances of the fractions at 350 pmol/head, respectively.
nm with the MULTI-340. Retention times of In order to elucidate whether or not ll-n'.v and
HPLC Analysis of Planarian Retinoids 945
fractions (2-8 min) in Figure 5a have peaks which
are much largerthan those in Figure 5b, indicating
"350 3 M350 that retinyl esters were mostly extracted by the
0.004 0.004
I I hexane extraction. Thus hexane extracted almost
all of the all-trans retinol along with retinyl esters
in the planarian tissues leaving ll-cis and all-trans
retinals.
10 min 10 min
DISCUSSION
As shown in Figure 1, retinoids extracted from
the homogenates of 18 bodies of the planarian
were composed of all-trans retinal (2.1 mol%),
all-trans retinol (12.6mol%) and all-trans retinyl
ester (85.2mol%). Retinyl ester is probably a
main storage form of retinoids in the planarian
body and seems to exist in the oil-droplets, which
emit the green-yellow fluorescence as seen in
300 340 380 420 Figure 2. It haslongbeen known thatvitamin A is
stored mainly as retinyl ester in the livers of a
Wavelength (nm)
Fig. 5. (a) HPLC chromatogram of hexane extract numbers of vertebrate species and that these re-
from freeze-dried sample of the planarian heads tinyl esters are present in oil-droplets of the liver
prepared as mentioned in Fig. 4. (b) HPLC chro- fat-storing cells [16]. The planarian has oil-
matogram of retinoids extracted by the oxime droplets in the fixed parenchymal cells [17]. Prob-
method from the residues after the hexane extrac- ably the planarian is capable of storing retinol in
tion, (c) Absorption spectra of peak fractions ester form in the parenchymal cells.
indicated by numbers 2 and 3 (solid lines) and
standard syn 11-cw and syn a\\-trans retinaloximes Thehexaneextractfromthefreeze-driedplanar-
(dotted lines). ian heads contained almost all of the all-trans
retinol and the retinyl ester in the tissues. How-
ever, all-trans and ll-cis retinals could not be
all-trans retinals were bound to any protein in the detected in the extract. Both of the retinals were
planarian tissues, we carried out an experiment as extracted from the residues, after the hexane ex-
follows. The hexane extract from the freeze-dried traction, as the oximes. It is well known that
samples of planarian heads was analyzed by retinalscombinedwith aminogroupofaprotein in
HPLC. As shown in Figure5a, the peak of all- tissues (e.g. vertebrate and invertebrate retinas)
trans retinol (peak 1) was quite large, while the are not extracted by hexane. Therefore the retin-
peaks due to ll-cis and all-trans retinals were not als in the planarian may be bound to an uniden-
found. Figure 5b is an HPLC profile of retinoids tified proteins in the tissues.
extracted from the residues by the oxime methods All-trans retinalwas detected in extractsofboth
after the hexane extraction. The figure indicates head and tail of 28 planarians (see Fig. 3), while
substantial peaks, numbered 2 and 3, correspond- ll-cis retinalwas detected in extractsderivedfrom
ing to syn ll-cis retinaloxime and syn alll-trans 600 planarian head pieces (see Fig. 4 and Fig. 5).
retinaloxime, respectively. Figure5c indicates the It is reasonable to infer that the ll-cis retinal was
relative absorption spectra of fractions corres- derived from the chromophore of visual pigment
pondingto peaks2 and 3 as curves 2 and 3. These of the planarian photoreceptor. The eye of the
spectra are very similar to those of standard syn planarian used in this experiment is assumed to be
ll-cis retinaloxime and syn all-trans retinaloxime asphere ofabout 90^m in diameter. The rhodop-
(indicated bydottedlines), respectively. The early sinconcentration ofinvertebrate photoreceptors is
946 K. Azuma, N. Iwasaki et al.
0.3-0.4mM [18]. We can estimate the amount of fishes. J. gen. Physiol., 25: 235-245.
the chromophore per planarian eye, if the eye is 8 Wald, G. (1957) The metamorphosis of visual
assumed to be filled with microvilli which contain systems in the sea lamprey. J. gen. Physiol., 40:
rhodopsin in the concentrations of 0.3-0.4mM. 901-914.
The calculated value of the amount of chromo- 9 Matsui, S., Seidou, M., Uchiyama, I., Sekiya, N.,
Hiraki, K., Yoshihara, K. and Kito, Y. (1988)
phore was 0.11-0.15pmol per eye, which was 4-Hydroxyretinal, a new visual pigment chro-
close to the amount of ll-cis retinal indicated in mophore found in the bioluminescence squid,
this experiment i.e., 0.11-0.19pmol per head. Watasenia scintillans. Biochim. Biophys. Acta, 966:
370-374.
10 Suzuki, T. and Eguchi, E. (1987) A survey of
ACKNOWLEDGMENTS 3-dehydroretinal as a visual pigment chromophore
in various species of crayfish and other freshwater
The authorsthankprofessorDr. T. P. Williams (Flor- crustaceans. Experientia, 43: 1111-1113.
ida State University) for critical reading of the manu- 11 Vogt, K. andKirschfeld, K. (1984) Chemicalidenti-
script and Messrs. H. Fujino, T. Tanaka, A. Yuda and tyofthechromophoresofflyvisualpigment. Natur-
M. Miyazaki for collections ofthe planarian worms.
wiss., 71: 211-213.
12 Spudich,J. L. andBogomolni, R. L. (1988) Sensory
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