Quantification of L-Dopa and Dopamine in Squid Ink: Implications for Chemoreception

9 Pages·1994·3.5 MB·English

by  M T Lucero

Reference: Biol- Hull 187: 55-63. (August, 1994)
Quantification of L-Dopa and Dopamine in Squid Ink:
Implications for Chemoreception
MARY T. LUCERO1 HERALDO FARRINGTON2 AND WILLIAM F. GILLY2
, .
1University ofUtah, Department ofPhysiology. 410 Chipeta Way, Salt Lake City, Utah 84108, and
2Stanford University, Hopkins Marine Station. Pacific Grove, California 93950
Abstract. Squid ink is an alarm substance that both that servesas an effective visual distraction for predators.
confuses predators and alertsconspecifics tothe presence Ink may also contain compounds that interfere with a
ofdanger. Although theejection ofink isapowerful visual predator'schemosensory abilities, although the behavioral
stimulus, studies also indicate a chemical component to evidence is largely anecdotal (Fox, 1976: MacGinitie and
the signal. Squid ink is composed mainly ofmelanin pig- MacGinitie, 1968). In support ofthese ideas, Prota et at.
ments, but the nonpigmented portion ofthe ink contains (1981) found a highspecificactivityoftyrosinaseinceph-
the enzymes and precursors of melanin synthesis. Our alopod ink and suggested that the in situ oxidation, by
previous behavioral studies showed that squid olfactory this enzyme, ofphenols in the ink to quinones may be a
organs detect L-dopa, a key chemical in melanogenesis. source ofthe hypothesized chemical messengers.
Squid olfactory neuronsalso respondtodopamine, abio- In addition to affecting predators, ink clouds may also
genie amine not previously described in squid ink. We alert conspecifics to danger, especially within the dense
performed HPLC on ink taken from the ink sacs ofadult schools ofmany squid species. Furthermore, thewarning
Loligo opalescens. The ink was conjugated with ortho- signal is likely to be chemical as well as visual because
phthaldialdehyde (OPA) and injected into the HPLC, and the visual component would be oflimited usefulness at
amine-containing compounds were detected fluoromet- night or in the dark ocean depths. In this respect, ink
rically. Standard curves constructed for L-dopa and do- would play a pheromonal role, because its release and
pamine allowed quantitation from individual ink sacs. detection could provide a basis for intraspecies commu-
We found that L-dopa was present in undiluted ink at a nication. Indeed, local application ofdiluted squid inkto
mean concentration of 1.15 mAI and was significantly the olfactory organ ofrestrained squid caused strong es-
greater than the mean dopamine concentration of cape jetting responses (Gilly and Lucero, 1992). In ad-
0.19 m.U. These values are greater than those at which dition. 1-3,4-dihydroxyphenylalanine(L-dopa),acolorless
both compounds are effective in behavioral and electro- compound found in cuttlefish ink (Jimbow et a!.. 1984),
physiological experiments. In addition we found that an elicited escape responses in the absence ofother metab-
unidentified antioxidant in the ink may prevent rapid olites ofmelanogenesis including tyrosinase. phenols, or
oxidation ofL-dopa and dopamine following dilution in quinones. Therefore, without rulingouteffectsoftheother
seawater. metabolites, initial behavioral experiments implicated L-
dopaasa potential chemical cue(Gillyand Lucero, 1992).
Introduction L-dopa is an intermediate, not only in the synthesis of
melanin and related pigments, but also ofdopamine and
Inking by cephalopods has long been recognized as an other neurotransmitters (Waite, 1992). Thus, preliminary
adaptive response to predation and physical threat. The physiological studieswere performed and showed thatdi-
ink clouds produced by these animals allow for retreat lutedsquid ink. L-dopa. anddopamineall inhibitedspon-
from a threatening situation and leave behind either a taneous firing in primary sensory neurons isolated from
diffuse "smoke screen" oracompacted, long-lastingdecoy squid olfactory organs (Lucero et a/.. 1992).
Because L-dopa and dopamine both mimic the effects
Received 9 November 1993;accepted 6June 1994. ofsquid ink on primary sensory neurons, we postulate
55
56 M. T. LUCERO ET AL
that ejected ink functions as a warning signal, and that (EDTA), 200 nAI: and heptane sulfonic acid, 1.2mA/.
the two metabolites are effector molecules. Neither pH 3.6. Biogenic amines in the ink weredetected electro-
substance, however, has ever been identified in squid chemicallywithan ESACoulochem (Model 5100A; 5021
ink. Therefore, we used high pressure liquid chroma- conditioning cell; 5011 analytical cell) in the reductive
tography (HPLC) to identify and quantify L-dopa and mode (Gomez-Nino el al, 1990).
We
dopamine inthe ink from individual ink sacs. report
here that squid ink contains free L-dopa and dopamine Standards andstandardcurvesforfluorescence
inconcentrationssufficienttoproduce physiological ef- detection
fects. Moreover, the ink also appears to contain an an- Because oftheirlow solubility and rapid oxidation, 50
tioxidant that retards the rapid oxidation ofthese com- nm L-dopa and dopamine standards were made up in a
pounds in seawater. solution of 10mm ascorbic acid and 150mm sodium
acetate(pH 10). Asanadded measuretopreventoxidation
Materials and Methods during preparation, all solutions were bubbled with N2
HPLCapparatus andfluorescence detection gasA,llplsacaemdploens iwcee,reanfdiltuesreedd w(0i.t2hi^nm6 hM.illipore) prior to
Chromatography was performed with two Waters derivatization, whichwascarried outbyadding25%.OPA
pumps connected to a Waters Model 720 System Con- by volume to the injection sample. The derivatized sam-
troller programmed to generate a dual-solvent, stepped- ples were then vortexed briefly and centrifuged (14,000
elution profile. PeaksweredetectedbyaFluoro-Tecdigital Xg) for 1 min. Volumes ofsupernatant, ranging from 20
filterfluorometerwithanexcitationwavelength of320 nm iul to 400 n\, were injected for analysis within 3 min of
and an emission wavelength of450 nm. Peak areas were derivatization. An example ofHPLC traces obtained for
integratedbyaWatersDataModule. Separation ofortho- 100-^1 injectionsof50^m L-dopaanddopamine isshown
phthaldialdehyde (OPA)-derivatized primary amineswas in Fig. 3A. Chemicals were obtained from Sigma (St.
performed on an Ultrasphere 3-Mm Octadecasilane (ODS) Louis, MO) and were HPLCgrade orACS reagentgrade.
mm mm
column of75 length and 4.5 internal diameter Standard curves (data not shown) wereestablished over
(Jaeckle and Manahan, 1989; Manahan, 1989). the range ofinjections that produced the best integrated
A combination of aqueous and nonaqueous solvents peaks. The L-dopa curve was based on 19 injections with
was used tocreate an elution profile with atotal run time injection volumes corresponding to L-dopa amounts
of 23 min. The aqueous solution (solvent A) contained ranging from 0.5 to 5.0 nanomoles. The dopamine curve
150mm sodium acetate (pH 7.2). 9.5% methanol, and wasbasedon 18 injectionswiththelowerlimitofdetection
0.5% tetrahydrofuran (THF). The nonaqueous solution at 1.25 nanomoles. Each curve was estimated by linear
(solvent B) was 100% methanol. regression, and the r values were 0.999 and 0.986 for L-
A modified stepped-elution profile was developed that dopa and dopamine, respectively.
allowed for the clear separation and signal integration of
L-dopa and dopamine at a flow rate of 1.8 ml/min. The Standards for electrochemical detection
time course for solvent B was as follows: constant 10% The standards used forelectrochemical detection were
cfornosmtantto210m%inf,rolimne4artionc9remaisne,toli2ne0a%r firncormea1setot4o m5i0n%, cmlauddeedfrLe-sdhopdaa,ilydoipnam1inNe,perdcihhlyodrriocxyacpihden(yPlCAac)etaincdaciind-
from 9 to 13.5 min, constant 50%. from 13.5 to 19 min, (DOPAC), and 3,4-dihydroxybenzylamine (DHBA).
linear increase to 90% from 19 to 21 min, lineardecrease DOPACisacatabolite ofdopamine that occursnaturally
to 10%' from 21 to 23 min. At 23 min. the initial condi- in squid ink. DHBA wasadded, asan internal control, at
tions were reattained and the column was ready for the the time ofextraction ofink samples being prepared for
nextinjection. TheelutiontimesforL-dopaanddopa=mine electrochemical detection (see below). Electrochemical
under these conditions were 7.82 0.08 (SD; n 19) detection ofthesefourstandards isexemplified in Figures
and 13.25 0.08 min (SD; /; = 18), respectively. 2A (50 ng each) and 2B (100 pg each). Standards were
run and calibrated at two different gain settings so that
Electrochemicaldetection the L-dopa and dopamine peaks from ink could each be
An additional HPLC column and detection method integrated at optimal sensitivities.
wHaPsLuCsesdeptoarvaetriiofnyroefsudlitlsutoebdtasiqnueiddwiintkhwtahseOpePrAfomremtehdodo.n Extraction andpreparation ofsquid inkforfluorescence
detection
a C-18 reverse phase column (Rainin) in phosphate-buf-
fered mobile phase consistingofmonosodium phosphate Live specimens of adult Loligo opalescens were col-
(Na-H2POj), 50 mA/: ethylenediaminetetraacetic acid lected in Monterey Bay, California. Squid were decapi-
L-DOPA AND DOPAMINE IN SQUID INK 57
tated, inksacswereremoved bydissection, andwhole ink each ink sample was 100 ^1, and the size ofthe resultant
was manually recovered. peaks guided the selection ofthe next injection volume.
An intact ink sac was placed on a sheet of Paranlm. Wheneverpossible, replicateswere injected at one partic-
mm
and the ink duct was transected about 5 anterior to ularvolumetoestablish a measureofinjection error. The
its emergence from the sac. A small glass rod was then standard curves for dopamine and L-dopa were used to
used to gently express the ink onto the Paranlm. Simul- transform the integrated peak areas from each analysisof
taneously, one end ofa sterile 10-^1 microcapillary tube ink into nanomolar quantities that were then corrected
was introduced into the emerging ink and allowed to fill for injection volume and dilution to obtain millimolar
halfway by capillary action. Several tubes were necessary concentrations that refer back to the original ink sample.
tocollect the ink from a single sac. Nitrogen gaswas used
to blow the ink out ofthe tubes and into a preweighed Quantification ofL-dopa anddopamine in ink by
Eppendorf vial that contained 1 ml of 150 mA/ sodium electrochemicaldetection
acetate and 10 mA/ ascorbic acid. The vial was immedi-
ately reweighed and put on ice. This procedure allowed Standards containing known amounts of L-dopa. do-
us to rapidly collect and weigh most ofthe ink within an pamine, DHBA, and DOPAC were separated on the
ink sac and simultaneously to dilute it into a solution HPLC,andpeakswereintegratedandcalibratedaccording
containing an antioxidant, thereby minimizing the risk to the known amount in the standard. The Waters Data
of oxidation. Only ink collections yielding more than Modulewasprogrammed to integrateandquantify peaks
15 mg ofink from a single squid were used in the quan- from ink samples having the same retention times as the
titative analysis ofL-dopa and dopamine. standards. The amounts ofbiogenic amines in each ink
The vial containingdiluted inkwascentrifuged (14,000 sample were corrected for oxidative loss through the alu-
Xg)for 10 minat4C. Theink supernatantwasremoved mina column by a factor of 3.13 relative to the internal
andcentrifuged in0.2 ^m filtertubesfor45 s. The filtered standard (DHBA). We were unable, however, to correct
ink supernatant was conjugated with OPA in the same for the loss that probably occurred between the time the
manner as described for the dopamine and L-dopa stan- samples were thawed and run on alumina columns and
dards above. the time oftheir injection into the HPLC.
Preparation ofinkfor electrochemical detection Results
In experiments where the electrochemical detection Identification ofL-dopa anddopamine in squid ink
bmeettwheoednwaasfoulsdedo,ftphaeraifniklmsawciwtahstbhleotitnekdddurcytaenxdtepnldaicendg corTrweospomnedtihnogdtsowLe-rdeopuaseadndtodiodpeantmiifnyethien HthPeLiCnk.peOankes
caionntndotatdihrneoipncsogllo0ef.c5tiinmokln tfeoulbfle.dtihTreehceatclpyeatriaantfteio/lamascnwoaribscaegtdeent1bl.uy5f-fpmerlrestsauenbdde, wwiatshttohcosoempoafrtehetshteanpdroafridlse,aannddetlhuetisoencotnidmewsaosfttohe'sppiekaek'
50 ^g/ml DHBA. After6sampleswerecollected, thetubes ttohetihenkibnky asadmdpilneg.4TnhaenoHmPoLleCstorfacLe-doofpfareasnhdidnkopfarmoimnea
werevortexed for 30 sand immediately frozen on dry ice
single ink sac diluted with the antioxidant ascorbic acid
Ufotrash.hipSmaemnptlefsrowmerMeontkeeprteyf,rCoazleinfoartnia-,7to0SCaltfoLrak3edCaiytsy,, ipsrescheodwenainndFfioglulroew1tAh.eSleavregrealL-sdmoaplal puenaikd.entTihfeiedsizpeesakosf
quicklythawed, vortexedbriefly,and run through alumina these smaller peaks in ink samples from different squid
silica columns (Gomez-Nino ct a/., 1990). L-dopa and
varied, as did the size ofthe dopamine peak.
catecholamines in the ink sample were eluted from the
4alduamyis.naThceolaulmunmiwniathpr1oNcedPuCrAe waansdnsetcoersesdaarty4toCrefmorov1e- in AFidgduirteio1nB.oTfhLe-daorpeaasaonfdthdeoLp-admoipnaeatnodtdhoepianmkinisespheoawkns
ascorbic acid from the ink because, as a reducing agent, were dramatically increased without affecting the overall
shapes or elution times ofany ofthe other peaks in the
ascorbic acid interferes with the oxidation-reduction re-
sample. Thus, the L-dopa peak in ink had an average elu-
action used in electrochemical detection (Gomez-Nino et tion time of 8.08 0.15 min (SD, /; = 47), and the do-
al.. 1990). pamine elution time averaged 13.49 0.26 min (SD, n
Quantification ofL-dopa anddopamine in ink by = 41). These values were very close to the elution times
for the L-dopa and dopamine as cited above. Similarly,
I'mtrescencedetectit i when preparations ofink and standards were run on the
Several different volumes ofa given sample were in- reverse phaseCl8 column anddetectedelectrochemically,
jected into the HPLC so that a range ofwell-integrated the ink samples had peaks that eluted at the same time
peaks could be obtained. The first injection volume for as the standards. In Figures 2A and C, the gain of the
58 M. T. LUCERO ET AL.
to
to
B
Squid Ink Squid Ink + LD + DA
t t t t
LD DA LD DA
Figure 1. Primary amines were identified in squid ink by 'spiking' with L-dopa (LD) and dopamine
(DA).(A)TheHPLCtraceobtainedby injecting50^1ofdilutedsquidinkconjugatedwithOPA(asdescribed
in the Methods) showsa prominent L-dopa peak at 7.86 min and a small dopamine peak at 13.4 min. (B)
50/jl ofa'spiking'cocktail containing250^1 ofdilutedsquid ink, 25 n\ ofL-dopa,and 25 jul ofdopamine
wasinjected intothe HPLCtopositively identifythe L-dopaanddopamine peaks. Both peaksincreasedin
areawith littlechange in elution time.
electrochemical detector was set to 100, and an L-dopa Quantitative analysis
peak can be seen at 4.72 min in both the standard and in
the ink. The peaks for dopamine and DOPAC in ink are After correcting for the injection volume and dilution
not resolvable at this gain setting, but a bump at 16- of the ink, the mean concentrations of L-dopa and do-
17 min is discernible. With the gain setting turned up to pamine were obtained for each ink sac analyzed by the
9900, thedopamine and DOPAC peaks in thesame sam- fluorescence method (Table I). The number ofinjection
ple ofink are clearly resolved (Fig. 2D), but the L-dopa volumes for a given ink sac (/;) is less for dopamine than
peak (4.74 min) is now saturating. As mentioned in the L-dopa. because several ofthe injection volumes used to
Methods, 50 /xg/ml DHBA was added to the ink as an obtain an L-dopa peak contained less than the minimum
internal standard andappearsasthelargesaturatingpeak detectable amount ofdopamine (1.25 nanomoles). Inte-
in Figures 2Cand 2Dat 10.37 and 10.34 min respectively. gration ofthedopamine peaksin suchprofileswasdeemed
Several amino acids and neurotransmitters were run unreliable, and the resulting valueswere not used for fur-
as controls, either with fluorescence or electrochemical ther analysis.
detection methods, totestwhethertheirelution timesdif- The concentration of L-dopa in all of the ink sacs
fered from those of L-dopa and dopamine. None ofthe analyzed with the fluorescence detector averaged 1.15
substances tested (aspartate, glutamate, arginine, tyrosine, 0.56 mUf(n = 10), and the dopamine concentration
taurine, phenylalanine, ammonia, ammonium, octopa- averaged 0.19 0.07 mAI (u = 9). The highest values
mine. histamine, and norepinephrine) overlapped with for each substance, approximately twice the means,
the L-dopa ordopamine peaks, but the retention timesof were found in ink sac G. The coefficient of variation
someoftheaminoacidsand aminesweresimilartothose (CV). or percent error, in measuring the L-dopa and
of other peaks in the ink sample, suggesting that these dopamine in individual ink samples by fluorescence
substances may also be present in ink. detection averaged 3% (CV = SD/mean X 100%). This
L-DOPA AND DOPAMINE IN SQUID INK 59
STANDARDS
SS
g s
t t tt t t tt
LD DHBA DOPAC DA LD DHBA DOPAC DA
SQUID INK
t ft t t tt
DHBA DOPAC DA LD DHBA DOPAC DA
Figure 2. Electrochemical detection verifies the presence ofL-dopa(LD) and dopamine(DA) in squid
ink.(A)TheHPLCtraceofa 10^1injectionofstandardsolutioncontaining500pg//jleachofDA,DOPAC
(dihydroxyphcnyl aceticacid), DHBA (3,4-dihydroxybenzylamine). and LDwasobtained atagain setting
of100.(B)Thestandardsolutionwasdilutedtoaconcentrationof10pg/M>andthe 10-^1injectionisshown
atagain settingof9900. The initial peaksatabout 2 min in all tracesrepresentthe solvent front reaching
thedetector.(C)TheHPLCtraceof20nIofsquid inkpreparedforelectrochemicaldetection(seeMethods)
recorded at the samegain asin (A) showsalarge L-dopapeak at4.72 minand asaturating DHBA peak at
10.37 min. The small DOPAC and dopamine peaks were not integrated. (D) The HPLC trace of 10 M! of
squid ink from the same sample asin (C) recorded at again of9900. shows that, although multiple peaks
arevisibleat thissensitivity, the L-dopa, dopamine, DHBA, and DOPAC peaksareeasily recognized.
60 M. T. LUCERO ET AL.
Table I
ConcentrationsofL-dopaanddopamineforink twin nulmdiialink
sacks
I-DOPA AND DOPAMINE IN SQUID INK 61
T = T= 10.5 h
LD + DA -f Ascorbate
B
LD + DA + Sea Water T = 68 mln
a
Squid Ink + Sea Water T = 93 mln
t t t t
LD DA LD DA
Figure3. HPLC tracesreveal that L-dopa (LD) anddopamme(DA)are stable in squid ink and in the
presenceofascorbicacid,butoxidizerapidlyinfilteredseawater.(A) 100^1of50pML-dopaanddopamine
in Na-acetatebuffercontaining 10mAfascorbicacidwasinjected intothe HPLCattimezero(a)andafter
10.5h at room temperature (b). (B) HPLC traces show rapid oxidation of200 jul of50 nML-dopa and
dopamineinseawaterattimezero(a)andafter68 minatroomtemperature(b).Thesmallsharppeakwith
an elution time of0.6 min was assumed to be an oxidation product or contaminant and was not further
characterized.(C)SquidinkdiluteddirectlyintoseawaterandinjectedintotheHPLCattimezero(a)shows
verylittleoxidation ofL-dopaanddopamineafter93 minat room temperature,despitegassingfor25 min
withO;(b).
essary to elicit behavioral (Gilly and Lucero. 1992) and that stabilizesthesechemical messengers in seawater may
physiological (Lucero et ai. 1992) responses and are be present in ink.
therefore physiologically significant. Our work thus sup- Both fluorescenceandelectrochemical detection meth-
ports the idea that ink is an alarm substance and that ods were employed, after HPLC separation, to identify
productsofmelanin synthesis(L-dopaanddopamine)are and quantify L-dopa and dopamine in squid ink. Both
present in the ink at levels that provide chemical signals methods showed peaks in the ink that eluted at the same
to other squid. In addition, the levels of these biogenic times and with the same characteristic shapes as L-dopa
amines varyconsiderably among individual ink sacs, and anddopamine standards. With fluorescence detection, the
this variability is not due to differential oxidation ofthe average concentrations for L-dopa were 1.15 0.56 mAf
inkduringhandlingortoerrorsin measurement. Finally, (n = 10), and for dopamine were 188 67 /uM ( = 9).
wepresentpreliminaryevidencethatanatural antioxidant L-dopa concentrations were also obtained by electro-
62 M. T. LUCERO ET AL.
chemical detection that were as high as the lowest levels ditions (Aime and Fasano, 1990; Crescenzi et ai, 1993).
measured with the fluorescence (ink sac I), and the max- This reductive capacity of melanin pigments has led to
imum dopamine concentration measured electrochemi- thesuggestionthat, in livingsystems, melanin playsarole
cally wasabout 3 time lowerthan the lowest value found in removing H:O: (Aime and Fasano, 1990). Although
with fluorescence. But oxidation experiments with stan- the melanin pigmentscould bestabilizingL-dopaanddo-
dards (Fig. 3A) shmowMed that, over a period of 10.5 h in pamine in squid inkbypreventingoxidation, anotheran-
the presence of 10 ascorbic acid, dopamine oxidized tioxidantmayalsoexist,becausetheexperimentsin Figure
more rapidly than L-dopa. Thus, the reduced concentra- 3 were run on ink from which the majority of melanin
tions of both compounds measured electrochemically pigments had been physically removed by centrifugation
probably reflect oxidation that occurs after the removal and filtration. Perhaps, in addition to the melanin, an
ofantioxidantswiththealuminasilicacolumn. Similarly, antioxidant such as ascorbic acid is released with the ink.
slow differential oxidation of L-dopa, dopamine, and In any case, the variability among ink sacs is not due to
DHBA may have occurred during handling, shipment, rapid oxidation and may depend on the length of time
We
and storage ofthe ink that cannot be corrected forby the that the ink wasstored in the ink sac. did not attempt
known amount ofDHBA degradation. to record the inking history of the squid used in these
TheconcentrationsofL-dopaanddopamine measured experiments, but some squid inked upon capture and
with the fluorescence detector are higher than those others did not.
needed to elicit chemosensitive behavioral responses from In summary, these experiments show that squid ink
intact animals (Gilly and Lucero, 1992) or electrophysi- contains high concentrations of L-dopa and dopamine.
ological responses from sensory neurons isolated from the This study supports earlier findings that these biogenic
squid olfactory organ (Lucero et a/., 1992). Exact quan- amines are behaviorally (Gilly and Lucero, 1992) and
titative comparisons are difficult because the dilution as- physiologically (Lucero et ai. 1992) relevant olfactory
sociated with pressure ejection from a small pipette is stimuli. Further work will be necessary to identify the
uncertain. Published experiments have revealed thatsquid antioxidant in the ink, determinethesourceofvariability
can detect a 1:20 dilution ofthe ink (Gilly and Lucero, between animals, and characterize the chemical message
1992), suggesting that the detection ofchemical stimuli in a natural ink plume.
by squid is quite sensitive.
Thus, even when ejected ink is diluted into seawater, Acknowledgments
these compounds will still be detectable by a squid, at
The authors thank Dr. Randy Alberte and Dr. Sal Fi-
least fora short time. The lifetime ofthischemical alarm
dsiogpnaalmiwnoeulodrpLr-edsoupmaabrleymdaeipnenbdouonndthteo etxhteenmtuctousw-hliickhe dHoPnLeCfoerqutihepimrengetnearnodsigtryateifnulallylaocwiknngowulsedtghee tuhseeionvfatlhue-
able assistance ofDr. Richard Zimmerman and Dr. Bruce
cthoempinoknemnatssstoofgtehteherinikntchoamtpaarceterdesfpoonrsmi.blAevfaolruahbolledianpg- Dinger. We also thank Dr. Jia Chen for running the alu-
mina silica columns and Taylor Liu for obtaining live
proach to the biological relevance ofour findings will be squid. We appreciate Jimmy Lucero's efforts both in
to employ electrochemical probe analysis of natural ink
poAfltueTtmmhheaees,dcto1oo9pnm8ace8em;anistnMureorarteogiritoechnesesttioegafmn/.pa.Llo-r1dai9nol8p9aea)jn.edacntsedpdatdiiaonlpkcahm(aiMranoceotrereviasratiinecdds wtFmeiaarnisaenlyftluayB,inandywieenfdogarbrtyephrgeOorfvasfitiqedcufieiundlgoftauoNnstadahvceaicnlfeisdsRsiheritsneoegctalciirvoncegmhsmtqGuhureniaidirn.ttdyTihNsoisOfseOcMwtOoioo1nrn4-k.-
89-J-1744.
greatly among ink sacs, but the ink sac with the highest
concentration of L-dopa also had the highest dopamine
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