physiological basis of stomach oil formation in leach's storm-petrel

13 Pages·2003·1.01 MB·English

PHYSIOLOGICAL BASIS OF STOMACH OIL FORMATION IN
LEACH'S STORM-PETREL (OCEANODROMA LEUCORHOA)
ALLEN R. PLACE,(cid:127) NINA C. STOYAN,2 ROBERTE . RICKLEFS2, AND
RONALD G. BUTLER 3
(cid:127)Centero f MarineB iotechnologUyn, iversityo f Maryland,B altimoreM, aryland2 1202U SA,
2Departmenotf BiologyU, niversityo f PennsylvaniPa,h iladelphiaP,e nnsylvani1a9 104U SA, and
(cid:127)Departmento f Scienceasn dM athematicsU, niversityo f Maine,F armingtonM, aine0 4938U SA
ABSTRACT.--Weex aminedt he compositiono f stomacho ils and the gastrointestinaal dap-
tations responsiblef or their forcmhaictikosn o ifn Leach'sS torm-Petrel, Oceanodromlae u-
corhoaW. e used tritium-labeledg lycerol triether (a nonabsorbableli pid-phasem arker) and
carbon-14l abeledp olyethylene glycol (a nonabsorbablea queous-phasem arker) to follow the
gastrointestinalt ransit of a homogenizedm eal fed to chicks.D ietary lipids remained in the
gastrointestinalt ract significantly longer (126 h mean retention time) than aqueousc ompo-
nents (12 h) due largely to different rates of gastrice mptying. Mean gastricr etention times
were 0.35 h for aqueouss olutionsa nd 70 h for neutral lipids. Stomacho ils from Leach's
Storm-Petrelc hicksa nd adults consistl argely (>90%) of neutral lipids (triglycerides,w ax
esters,a nd glycerol ethers). The compositioni s dynamic,a nd componentsa re partitioned
betweena n oil phasea nd aqueousp haseb asedl argely on water-lipids olubilities.T he paucity
of fatty acidsa nd phospholipidsi n stomacho ils derivesf rom limited solubility in neutral
lipidsa nd rapidg astrice mptyingo f polarl ipid constituentsP. roventricularli polysiso f neutral
lipids is low (<3%) and we found no evidenceo f proventricularl ipid secretionD. uring the
week or two before fledging, Leach'sS torm-Petrelc hicksr egulate the amount of stomacho il
by restricting the rate of gastric lipid emptying. Received2 0 January1 989, accepted1 0 June
1989.
THE ORDERP rocellariiformes is entirely pe- of the gastrointestinal tract in petrels slowed
lagic. Most speciesfe ed on lipid-rich prey, and the drainageo f oil into the intestine.T he denser
adults may commute long distancesb etween food and aqueousm aterialst end to separatea s
breeding sitesa nd feeding areas.W ith the ex- a bottom layer that blocks the accesso f oils to
ception of diving petrels (Pelecanoididae), all the pyloorpice ning. Those speciest hat regur-
Procellariiformesp ossessst omacho ils (Warham gitate stomachc ontentsa s a defense (e.g. ful-
1977, Jacob 1982). These oils are found in chicks mars) eject the lighter oil first, and they eject
and adults, breeders and nonbreeders, in birds solid or partly digestedm aterial only after the
captured at sea or at breeding colonies (Jacob oil supply is exhaustedR. ecents tudieso n gas-
1982). The ability to concentrated ietary items tric motility in Leach's Storm-Petrel chicks
into high-energy mealst hat occupys mallerv ol- (Duke et al. 1989) and on gastrointestinalp as-
umes and have lower osmoticl oads may pro- sageo f dietary lipids and aqueousc omponents
vide an advantage for both adults and chicks. in Gentoo Penguins (Pygoscelisp apua) and
Stomacho ils are important to the breeding ecol- southern Giant Petrels (Macronectegs iganteus)
ogy of these pelagic seabirds( Ashmole 1971). (Roby et al. 1989) establishedt hat aqueousd i-
Nearly 80% of the energy delivered to chicks
of Wilson's Storm-Petrel (Oceaniteso ceanicusr)e - ventriculuso f procellariiformsw hereasd ietary
sidesi n stomacho ils (Obst 1986) and 30% of the neutral lipids are retained.M oreover,m ean re-
energy metabolized by incubating Leach's tention times in petrels for lipid phase (20 h)
Storm-Petrels (Oceanodromlae ucorhoar)e sidesi n and for aqueous phase (10.7 h) were signifi-
stomach oils (Ricklefs et al. 1986). cantly longer than in penguins( 8.9 h for lipid
Cheah and Hansen (1970b) thought that oils phase,a nd 7.6 h for aqueousp hase)( Roby et al.
accumulatei n the stomachb ecausel ipids are 1989).
digested more slowly than proteins. Warham We describe the mechanisms and kinetics of
(1977)s uggestedth at the physicala rrangement stomacho il formation in a representativep ro-
687 The Auk 106: 687-699. October 1989
etary componentse mpty rapidly from the pro-
688 PLAC(cid:127)rE AL. [Auk,V ol. 106
cellariiform, Leach's Storm-Petrel. We relate the ony on nearby Little Duck Island. Depending on the
mechanicso f stomacho il formation to the pro- experiment,c hicksw ere either taken to the laboratory
cessb y which procellariiformsa re able to use for feeding experimentsa nd then returned at the ex-
periment'sc onclusiono, r they were fed labeledm eals
the dietary lipids. We show how birds concen-
in the field and then replaced in their nest burrows
trate food into a store,t he compositiono f which
(wheret hey remained)A. ll the chicksf rom Little Duck
is determinedl argelyb y propertieso f the meals,
Island were of known age. Other studiesw ere con-
the lipids, and a specialized gastrointestinal
ducted in 1986 at the Bowdoin Scientific Station on
morphology. Kent Island, New Brunswick, just south of Grand
Manan Islanda t the mouth of the Bayo f Fundy.C hicks
MATERIALS AND METHODS were removed from burrows and kept in a laboratory
on the island until the conclusiono f the study. The
Radiolabelasn d fiuors.--Tri[1-(cid:127)4C]olein (122 mCi/ ageso f Kent Island chicks were estimatedf rom mea-
mmol), L-[(cid:127)4C(U)]proline (266.4 mCi/mmol), [1-(cid:127)4C] surementso f wing cord (Ricklefs et al. 1985).
L-c(cid:127)-dioleoyl, [dioleoyl- 1-(cid:127)4C]-phosphatidylcholine Diet of captives ubjects.--Duringla boratorye xperi-
(114 mCi/mmol), and L-c(cid:127)-l-palmitoyl-2-oleoyl- ments, chicks were fed freeze-dried krill (Euphausia
[oleoyl-l-(cid:127)4C]-phosphatidylcholine( 52.6 mCi/mmol) superbaa) nd freeze-dried copepods( predominantly
from New England Nuclear (Boston,M assachusetts), Limnocalanums acrufus8, 7% of biomass).A 20% (w/v)
[1-(cid:127)*C] cetyl alcohol (24 mCi/mmol), D-[(cid:127)*C(U)] glu- homogenateo f either of these items was made with
cose( 4.28 mCi / mmol), n-[1-(cid:127)*C]-hexadecane (61 mCi / water and supplementedw ith either 35% (w/w) olive
mmol) from Amersham( Arlington Heights, Illinois), oil (triglycerides upplement)o r 35%( w/w) cetyl oleate
[1-(cid:127)4C] stearic acid (52 mCi/mmol) from Research (wax supplement). Nightly meals of 5-8 cc (ca. 88-
ProductsI nternational (Mount Prospect,I llinois) were 141 kJ/meal) were delivered with a disposable5 -ml
found to have radiopurities > 98%b y thin layer chro- syringe attachedt o a 10-cm length of polyethylene
matography. We used [1,2-3H] polyethylene glycol tubing insertedt hrough the esophagusin to the stom-
(M.W. 4000, 1.4 mCi/mmol) from New England Nu- ach. All chicksa cceptedm ealsw ithout regurgitation
clear (Boston,M assachusettsa) nd [U-(cid:127)4C] polyethyl- and exhibited normal development, based on daily
ene glycol (M.W. 4000, 15 mCi/g) from Amersham measurementso f wing growth.
(Arlington Heights, Illinois) without further purifi- Monitoringa queous-lippidh asep artitioning.--Ourb a-
cation. Scintillation fluors were ACS II (Amersham, sic strategy was to monitor the movement of carbon-
Arlington Heights, Illinois) and BiosafeI I (Research 14 labeled dietary constituentsi n relation to tritium
ProductsI nternational,M ount ProspectI,l linois). Du- labeled aqueous-phasea nd lipid-phase markers. In
plicates amplesw ere countedt wice on a BeckmanL S such studies it is important that each marker have
3801 scintillation counter, and a quench correction similar propertiest o componentso f the phaseu nder
curve was derived for different extracts and tissue study( Wigginsa nd Dawson 1961;C arlsona nd Bayley
types using an H number calibration. The counting 1972ab, ). Polyethyleneg lycol( M.W. 4000)i s a widely
efficiencyf or (cid:127)4C in the samplesv aried from 88.0%t o accepteda queous-phasem arker (Wingate et al. 1972)
75.4%, and that for 3H from 24.0% to 11.0%. The coef- and glycerol triether is a suitable neutral lipid-phase
ficient of variation for replicates amplesa veraged3 .0 marker (Morgan and Hofmann 1970; Carlson and
+ 1.3% (ñSD) for tritium and 1.9 + 0.9% for carbon- Bayley 1972a,b ; Meyer et al. 1986; Roby et al. 1989).
14. All radioactivities are expressed in /(cid:127)Ci (1.00 Thesec ompoundsa re nonabsorbablen, ontoxic,n on-
microCurie = 37.0 kilobequerals). degradable by digestive or bacterial enzymes, and
Tracera nd carrierw ax esters ynthesis.--Labeledw ax they do not influence the normal absorptiono f di-
ester( [1-(cid:127)4C] cetyl oleate) and carrier cetyl oleate was etary aqueousn utrients or fat.
synthesized( Placea nd Roby 1986).T he radio purity To test the phases pecificityo f eachm arker, several
(2.2 mCi/mmol) was 98.7%,t he overall yield ranged sampleso f stomacho il and excretaw ere subjectedt o
from 45% to 75%. The chemical purity was >98%. Bligh and Dyer extraction( 1959).O f the *H-GTE ra-
Glycerol triether [1-(9 cis-octadeceny2l), 3 didodecyl dioactivity, 93 ñ 1.2% (n = 4) was recoveredi n the
glycerolt riether] wass ynthesizeda sd escribed( Mor- chloroforml ayer while 96 ñ 2.5 % (n = 4) of (cid:127)4C-PEG
gan and Hofmann 1970) by BACHEM. The tritiated radioactivity appeared in the aqueousp hase. Thin
triether (3H-GTE) was prepared by reduction with layer chromatographyo f both excretedm arkersi n-
platinum asa catalyst( New EnglandN uclear,B oston, dicatedt hat neither had been substantiallym etabo-
Massachusettsa) nd purified (Roby et al. 1989). Carrier lized (i.e. radiopurity of each was at least 95%o f that
triacylglycerowl as purified from olive oil (Placea nd prior to ingestion).
Roby 1986). Samplingp roventriculusc ontents.--We obtained
Study areas.--Laboratory experiments were con- stomachs amplesw ith a 5-ml syringeu sed to gently
ducted in 1986 and 1987 at the Mt. Desert Island Ma- suckc ontentsth rougha 10-cml engtho f polyethylene
rine BiologicaLl aboratoryo n Mt. DesertI sland,M aine. tubing from the esophagusi nto the proventriculus
Storm-petrelc hicksw ere obtainedf rom a nestingc ol- (Grubb 1971). Stomach oils were separated from the
October1 989] StomachO il Formation 689
aqueousa nd particulate phasesb y centrifugation at fed 3 ml of a 1:1 (v/v) mixture of purified olive oil
10,000 x g for 10 min. and cetyl oleate,w ith glycerolt ri [9,10 (n)-(cid:127)H]oleate
Gastrointestintraal nsitt imesfo r lipida nda queoucso m- (12/zCi/ml) added. Aliquots were taken after 15 rain,
ponents.--Fousrt orm-petrecl hicks( age= 33-37 days, and the chicks were returned to their burrows with
mass= 58 + 4.98g ) were fed a radioactivelyta gged, the entrancesb lockedt o prevent adultsf rom return-
homogenizedm eal (2 g) of calanoidc opepodsW. e ing with food. After 24 h, the chicks were fed 3 ml
addedt he nonabsorbablew ater solublem arker [1-(cid:127)4C] of 1:1 (v/v) purified olive oil and cetyl oleatew ith
polyethyleneg lycol( M.W. 4000,P EG)a nd 50 mg/ml labeled glycerol tri [1-(cid:127)4C]oleate( 0.3/zCi/ml) added.
of unlabeledP EGt o markt he aqueousp hase( 1.3/zCi/ Samplesw ere removeda fter 15 min. We usedl abeled
meal). To label the lipid phase,w e added (3.7 /zCi/ triglyceridest o estimate lipid volume becausel ittle
meal) [3H]-labeledg lycerolt rierher (1-octadecyl-2,3- (<1.0%) lipolysiso ccursin the proventriculusfo r up
diodecyl glycerol, GTE). The ratio of [3H]/zCi/(cid:127)4C/zCi to 36 h (Placea nd Roby 1986).
of the food was 2.83 ñ 0.08 (n = 4). The meals were We calculatedt he apparent volume of aqueouso r
delivered by tube and syringe. After feeding, each lipid componentso f the proventriculus(, V) at time t
chick was placedo n a polyethylenem eshp latform from marker dilution by the expression( V(cid:127)C,/C(cid:127)) -
suspendedin a 2-galp olyethylenec ontainert o collect V,,w here V(cid:127) is the analyzeds amplev olume,V , is the
excreta.C ontainersw ere kept in the dark and main- volume of the marker fed, C(cid:127) is disintegrations/rain
tained at 14 ñ 3øC which simulated the nest environ- (DPM) in the samplev olume, and C, is the DPM in
ment. Chicksw ere periodicallyt ransferredt o clean the fed solution( Diamonde t al. 1986).G astrice mp-
containers, and the excreta was collected. Unlabeled tying was modeled using the exponentialf unction
meals( 5-8 g) were fed nightly to chicksA. ccumulated (Stubbs 1977, Smith et al. 1984),
excreta in each container were extracted with 50 ml
of chloroform:methanol( 2:1) and filtered on glass V(cid:127) = V, x e-(cid:127)'xb(cid:127) (2)
fiber filters to obtain a single phasee xtract.A liquots
(5 ml) were placed in scintillation vials and the sol- where V(cid:127) is the gastric volume at time t, V, is the
vent was removedu nder nitrogen evaporation.F luor initial volume fed, and b is the exponentialr ate of
was added and the vials counted. Food retention time emptying.
was estimatedu sing the exponentialf unction: Gastricli polysios f stomacohi ls.--Six chicksf rom Kent
Island( age= 68.6 ñ 3.14d ays,m ass= 73.5 ñ 12.5g )
nt = bo - eI -blXl(cid:127)-b2(cid:127) (1) and 4 chicksf rom Little Duck Island (age = 58 + 3.5
days, mass= 64.3 ñ 2.5 g) were fed 2 g of lipid
where n, is the proportion of marker excretedf rom containing 12.5 (cid:127)Ci of [(cid:127)H-GTE]-marker and 5/zCi of
initial administrationto time t (Hove 1984).T he pa- [(cid:127)C]-lipid. The carbon-14la beledl ipids were either
rameterb 0i s the asymptoticre coveryo f marker,b (cid:127) is glycerolt ri [1-(cid:127)C]oleate in olive oil or [1-(cid:127)C]cetyl
the rateo f excretion(p erh our),a ndb 2i s the delayi n oleatei n cetyl oleate.A fter 15 rain, a sampleo f stom-
hours before marker is recovered. The total mean re- ach oil was taken and the excreta collected. An ad-
tention time (TMRT) is the sumo f b2a nd the recip- ditional stomacho il samplew as taken 24 h after feed-
rocal of b(cid:127). Estimates and 95% confidence intervals for ing. Aliquots of stomach oils were counted for dual
thet hreep arameterwse reo btainedb y nonlinealre ast label radioactivitya s describeda bove.
squaresre gressionw eightedb y the varianceo f rep- As a more sensitivet esto f differentiall ipolysiso f
licates (Johnson et al. 1981). waxesa ndt riglycerides,6 chickso n Little Duck Island
Aqueouasn d lipid emptyingra teso f the proventricu- (age = 42.3 ñ 1.37 days,m ass= 66 + 9.25 g) were
lus.--To calculateth e contributiono f proventriculus fed 3 ml of 1:1( v/v) mixtureo f purifiedo live oil and
emptying time to transit time, we used a double-iso- cetyl oleate with glycerol tri [9,10 (n)-(cid:127)H]oleate (7.7
tope dilution technique (Diamond et al. 1986). To /zCi/ml) and [1-(cid:127)C]cetyl oleate (0.14/zCi/ml) added.
measurea queouse mptying rates,8 chicks( age = 64.1 Samplesw ere taken after 15 rain, and birds were re-
ñ 5.0 days, mass= 71.3 ñ 10.0 g) were first fed 5 ml turned to their burrows. The chicks were removed at
of an isotonic( 300 toOsin/l, accordingt o Schmidt- 4, 24, and 48 h after feedingf or further samplingo f
Nielsen [1960]) glucose/water solution labeled stomach oils. The chicks were allowed to receive meals
with(cid:127)4C-polyethyleneg lycol (50 mg/ml PEG; 0.017 from their parentsA. liquotsw ere analyzedf or radio-
/zCi/ml). After 5 rain (t = 0), an aliquot was removed activity and chemicalc omposition.
and dilution of label determined. After 1, 2, 4, or 8 Stomacho il composition.--Samplwese re taken from
h, we fed 2 ml of an isotonicg lucose/watesr olution 12 Kent Islandc hicks( age= 66.2 + 3.52d ays,m ass
labeledw itha secondm arker(cid:127)H, -polyethyleneg lycol = 62.0 ñ 5.75 g) that had been removed from their
(50 mg/ml PEG; 0.071 /zCi/ml). Again, 5 rain after burrowsa nd kepti n the laboratoryfo r 24 h. We with-
each feeding, an aliquot was removed and dilution drew stomacho il from theseb irds 5 rain after they
of the label determinedT. o measuret he emptying were fed labeledg lucoseo r proline,a nd then again
rateo f lipid componentisn the proventriculus6, chicks after 24 h. We alsot ook samplesfr om randomlys e-
(age = 48.5 ñ 4.6 days, mass= 76.3 ñ 9.65 g) were lectedc hicks( age= 50.3 _+1 .42d ays,m ass= 80.3 ñ
690 PLACEET A L. [Auk,V ol. 106
7.20 g) on Little Duck Island for stomacho il com- ach oils and excretab y thin layer chromatography
position analysis. (TLC). Aliquots containing equivalent counts were
Field observationsin dicatedt hat chicksr etain sig- spottedo n the preabsorbenat rea of channeleds ilica
nificant( 5-8 ml) quantitieso f stomacho il during pre- G plates( Uniplates,A naltech),d evelopedw ith hex-
fledgingw eight loss.A n experimentw as performed ane: diethyl ether: acetica cid (80:20:1),a nd scanned
with 6 chicks( age = 62 _+0 .95 days,m ass= 66 + 2.44 with a BioScan 100 radiometric scanner. This solvent
g, wing cord = 158 ___1 .7 mm) to determine whether systemr esolvesw ax esterst, riacylglycerolsf,a tty acids,
stomacho il compositiona nd volume might be reg- fatty alcohols,1 ,3-diacylglycerols1,, 2-diacylglycer-
ulated in older chicks. The 6 chicks were fed a meal ols, monoacylglycerolasn, d complexl ipids,i n order
of 20%( w/w) homogenizedk rill containing3 5%( w/ of decreasinrge lativem obility( Rf).T o separatec ho-
w) of a 1:1m ixture of cetyl oleatea nd olive oil. [(cid:127)H]- lesterole stersf rom wax esters,d ouble development
GTE (0.48 (cid:127)Ci/g) and tri [1-(cid:127)4C]oleate( 0.16 (cid:127)Ci/g). with hexane: diethyl ether (98:2) was necessary
Stomacho il was sampled 15 min later. Chicks were (Christie1 982).T he carrierg asu sedi n the radiometric
placed in polyethylene containerst o collect excreta. scanningw asP -10 (90%a rgon,1 0%m ethane)a t a flow
On the subsequent6 nights, the chicksw ere fed 3 g rate0 .5-1.01/min. The spatialr esolutionf or eachs can
of unlabeledh omogenizedk rill containing3 5%( w/ wass eta t 4 mm. The distributiono f label amongl ipid
w) olive oil. Beforef eeding,w e took stomacho il sam- classesw as estimated by integration of the counts
ples for lipid and radioactivitya nalysisw, eighed the under each peak after subtractionf or background.
chicks,a nd measuredw ing cords.A n additional ex- Theo verallc ountinge fficiencyfo r (cid:127)4C averaged1 0.5%
periment was conductedw ith 7 chicks that would whereast he countinge fficiencyfo r 3H averaged0 .5%
have fledgedi f not in captivity. acrostsh e plate.L abeledc etylo leatec, holesterool leate,
Polar and nonpolarl ipid repartitioning.--Labeled triolein, oleic acid, and cetyl alcohol were used as
phospholipids, L-oe-12-palmitoyl-2-oleoyl-[oleoyl-1-s tandardsto determinet he R! of thesem ajor lipid
(cid:127)4C] phosphatidylcholinea nd L-oe-dioleoy[ld, ioleoyl- classes.
1-(cid:127)4C]p hosphatidylcholinew, ere usedi n two separate Quantificatioonf lipidsu singT LC-fiameio nizationd e-
experimentso n phospholipidg astrice mptying.W e tection.--Operatingc onditionsf or the IatroscanT H-
added5 (cid:127)Ci of labeled phospholipida nd 50 (cid:127)Ci 3H- 10 analyzer (Iatron Laboratories,T okyo) were as de-
GTE to 49.5 g of homogenizedc opepodsW. e used 6 scribedb y Rigler et al. (1983). Samplesw ere spotted
birds (age = 42.2 __1_. 47d ays,m ass= 76.7 __1_4 .18g ) on type S-II chromarods( Iatron Laboratories)w hich
for eache xperiment;a nd we fed eachb ird a 5-ccm eal. were activatedb y scanningt hrougha hydrogenf lame
We withdrew samples5 min after the meal was ad- twice. Integration was performed by a Hewlett-Pack-
ministered, and 1, 3, 5, 24, and 48 h thereafter. The ard 3390-A integrator (Avondale, Pennsylvania).
5-min samplep rovided an initial ratio of the labels Chromarodws ere developedin equilibratedfi lter pa-
for comparisonw ith subsequenrt atios. Becausee n- per-lined glass tanks containing 75 ml of solvent
dogenousp hospholipasea ctivityw as high in the ho- (Harvey and Patton 1981,K aitaranaa nd Ackman 1981,
mogenized copepod meal, we repeated the experi- Parrisha nd Ackman1 983).S amplesw ere prefocused
ment using the same labels added to pure olive oil. with acetone.S ampleso f stomacho il were filtered,
In these experiments,t he ratio of labeled phospha- centrifuged,a nd 10 (cid:127)1 was added to 500 (cid:127)1 of chlo-
tidylcholine to glycerol triether was 5:1. Eachc hick roform. Total lipid content was determined by de-
(age = 45 __3_. 95 days,m ass= 80.2 __1_2 .3 g) was fed veloping the rodsi n chloroform:m ethanol( 1:1,v /v)
3 ml of labeledl ipid, and proventriculars amplesw ere along with a lipid standard.T he solvent front was
taken at 1, 2, and 3 h. run to 2 cm, and quantificationw as performed by
Six chicks( age = 45.5 + 2.5 days,m ass= 77.2 _+ TLC-FID. Lipid quantitiesw ere determinedb y com-
4.9 g) were fed 5 ml of a homogenizedc opepodm eal parison with a standard curve for concentrationso f
containing[ 1-(cid:127)4C] stearica cid (0.31 (cid:127)Ci/ml) and 3H- olive oil between 1 and 20 (cid:127)g. For determination of
GTE (1.85 (cid:127)Ci/ml) to determinef atty acid emptying stomacho il lipid compositionsr,o dsw ere developed
rates.S tomacho il samplesw ere taken at 5 min, 2 h, in one of severals olvents ystemsT. he first was 85 ml
5 h, and 24 h. To test whether nonpolar lipids (i.e. hexane: 15 ml diethyl ether: 0.1 ml formic acid for
alkanes)a ccumulatein the proventriculusw, e fed 6 50 min. Phospholipidc ompositionw as determined
chicks( age = 41.3 __1_. 03d ays,m ass= 70.5 _+8 .12 g) with double sequentiald evelopment. Rodsw ere first
0.5 ml of a copepodm eal labeledw ith n-[1-(cid:127)4C] hexa- developedf or 50 min in the aboves olvent,d ried in
decane (1.16 (cid:127)Ci/ml) and 3H-GTE (1.74 (cid:127)Ci/ml), and the oven, and burned halfway. Theser odsw ere then
we withdrew aliquotsa t 5 min, 24 h, and 48 h. developeda secondt ime with 75 ml CHC1(cid:127):35 ml
Meal and stomacho il samplesw ere centrifugedf or MeOH: 3.5 ml H20. Finally, samplesw ere analyzed
10 min to separatea queousa nd lipid phasesb efore for glycerole ther componentus singa doubled evel-
we removed aliquotsf or scintillationc ounting and opment system.S amplesw ere prefocusedt wice in
lipid analysis. acetonea, nda ir-driedb etweene achp refocusingR. ods
Tracerr ecoverya nd distribution.--Wed etermined la- were then developedi n hexane:diethyl ether:for-
bel distribution among various lipid classesin stom- mic acid (99:1:0.05) for 25 min, air-dried for 5 min,
Octobe1r 989] StomacOhi l Formation 691
and then redeveloped for 20 min. After drying at 100'
1000C for 5 rain, the rods were burned to a point
behind the wax esters.A fter this partial burning, rods
were dried and then developed for 40 rain in hexane:
diethyl ether: formic acid (80:20:0.1)t;h ey were then
burned the entire length of the rod. A standardc urve
wasg eneratedf or eachm ajorc lasso f lipids observed
(wax estersd, ialkyl glycerole thers,t riglycerides,f atty
acids, fatty alcohols,m ono- and diglycerides,a nd ; 40
phospholipids).
Enzymaticd eterminationo f lipid components.--Cho-
(cid:127). 2o
lesterol concentrations were determined with cho-
lesterol oxidase (Sigma Diagnostics,P rocedure No.
351). Triglyceride concentrations were determined 0 2'0 4'0 6'0 8'0 1(cid:127)0 12 0
using glycerol phosphate oxidase after lipase pro-
ductiono f free glycerol( SigmaD iagnosticsT, riglyc-
HoursP ost Ingestion
eride [GPO-Trinder] Procedure No. 338).
Fig. 1. Time courseso f gastrointestinale mptying
Statistics.--Resultasr e expresseda s means + stan-
of aqueous( I) and lipid (0) phasesi n 4 young (34-
dard error of the mean (œ+ SEM); n representst he
day-old) Leach'sS torm-Petrelc hicks.T he lines rep-
number of measurementsC. omparisonst hat involve
resentt he fitted exponentialf unction (Eq. 1) that best
percentagesw ere performed on arcsin transformed
describedt he rate of gastrointestinale mptying.
data. Differencesw ere considereds ignificant when P
-< 0.05, except for comparisonsi nvolving ratios of
When 65-day-oldc hicksw ere fed an identical
disintegrations/minute (DPM) of two markers;i n this
case, P -< 0.001 was chosen. In no case were fewer meal, 86.0 + 0.34% (n = 12) of the aqueous
than 1,000 DPM/sample observedf or either isotope. markerw as recovereda fter 24 h while only 1.8
Lines were calculated by a nonlinear least squares + 0.2% (n = 12) of the lipid marker was re-
iterative procedure( modified Gauss-Newtonm ethod) covered.T he unexcretedli pid markerw asf ound
(Johnsone t al. 1981). Parametere stimatesa re given in stomach oils. Older chicks with substantial
with their 95% confidence limits in brackets. Other
stomach oil (i.e. 9.1 + 1.5 ml, n = 6) excrete
statisticalp roceduresu sed are identified in the text.
significantly more lipid marker (31.4 + 7.2%,n
RESULTS = 6). This higher excretion rate wes accom-
panied by a low lipid assimilation efficiency
Gastrointestintaral nsitt imesfo r lipida nda queous (46.1 + 1.88%, n = 6) as comparedw ith 94 +
components.--Theex cretionc urvesd iffered for 0.35%, n = 12) (Place and Roby 1986, Place et
lipid and aqueousm arkers( Fig. 1). The aqueous al. 1986, Roby and Place 1986). The unassimi-
marker was excreteds ignificantly faster than lated lipids are excretedl argely unhydrolyzed
the lipid marker. By 24 h, 83.7 + 1.8% (n = 4) in older chicks.
of the aqueousm arkerw as recoveredc ompared Aqueousa- nd lipid-phasee mptyingr ateso f the
with only 11 ñ 1.1% (n = 4) of lipid marker. proventriculus.--Initiaal queousp roventricular
After 4.5 days,n early all of the aqueousm arker volumesf or eight 65-day-oldc hicksv aried from
was recovered (94.3 ñ 0.9%, n = 4) while only 0.9-5.1 ml. After 1 h, the gastrica queousv olume
44 + 2.1% (n = 4) of the lipid marker was re- decreased to 0.65 + 0.19 ml. We assume this
covered.A major portion (38 + 2.4%, n = 4) of indicated a proventriculuse mpty of aqueous
the unrecoveredl ipid marker residedi n stom- componentsT. he proventriculuss till contained
ach oils aspiratedf rom the proventriculus. 3.42 + 2.09 ml of stomach oil. The average
The fitted lines (Fig. 1) best describet he rate aqueousv olume emptied was 6.6 + 1.2 ml, and
of gastrointestinael mptying accordingt o Equa- the mean emptying time was 0.35 h. When the
tion 1. The timef or firsta ppearance(b (cid:127)) of either volume of aqueousm aterial in the proventric-
marker was similar: 1.4 h [0.4, 2.5] for lipids and ulus exceeded8 ml, emptying was not complete
0.9 h [0.7, 1.1] for aqueousc omponents.T he rate by 1 hour.
of passag(eb (cid:127)) for aqueousc omponentsw as0 .09. Gastrice mptying of lipid was significantly
ßh -(cid:127) [0.08, 0.10] while that for lipids was 0.008' slower. The initial stomach oil volume was 1.9
h -(cid:127) [0.007, 0.009]. The total mean retention time + 0.5 ml (n = 6). After 24 h, the volume was
(TMRT) was 12.0h for aqueousc omponentsa nd reduced to 1.5 + 0.2 ml (n = 6) with a mean
126 h for lipid components. emptying time of nearly 70 hours.
692 PLACEE TA L. [Auk, Vol. 106
TABLIt1 . The [3H]-GTE to (cid:127)4C-labeled lipid ratio in TABLE2 . Stomach oil composition (as weight per-
stomach oils from Leach's Storm-Petrel chicks 24 h centages)o f Leach'sS torm-Petrel chicksf rom Kent
after feeding. Island and Little Duck Island.
Kent Island Little Duck Island Little Duck
(n = 4) (n = 6) Kent Island Island
Lipid (n = 18) (n = 12)
Initial ratio Final ratio Initial ratio Final ratio
Wax ester 5.8 ñ 0.6 28.6 + 3.5
2.8 + 0.03' 2.9 + 0.006 2.5 ñ 0.02 2.5 ñ 0.07
Triglyceride 65.6 + 2.0 35.9 + 3.3
3.1 + 0.02 (cid:127) 3.2 _+ 0.114
Glycerol ether 0.8 + 0.2 28.7 + i0.6
Wax ester supplemented diet. Fatty acid 6.3 ñ 0.3 1.8 ñ 0.18
Triglycerides upplementedd iet. Cholesterol 9.5 + 0.9 2.0 + 0.2
Polar lipid a 12 + 0.6 2.9 ñ 0.43
Predominantlym onoglycerides.
Gastricl ipolysiso f stomacho ils.--Becauset he
tritium label resided in a nonmetabolizable, determined enzymatically (0.89 ñ 0.01 M, n =
nonabsorbablel ipid marker while the carbon- 10). We believe the differencei s due to glyceryl
14 label residedi n neutral lipid (triglyceride or ether diesters which co-migrate with triglyc-
wax ester),l ipolysisw ould lead to an increased erides in our solvent systema nd which are not
[3H]/[14C]r atio as neutral lipid was hydrolyzed assayede nzymatically. The organic phosphate
and carbon-14 label emptied from the proven- content of oil indicated a phospholipid level of
triculus.T here was no significantc hangei n the 0.29 ñ 0.26%( n = t0). The relative proportions
ratio after 24 h on either Kent Island or Little of eachl ipid componenti n stomacho ils did not
Duck Island (Table 1). changes ignificantlya fter 24 h (i.e. in a repeated
Similar results were recorded when chicks measures ANOVA, the amount (gg) of each
received mealsf rom their parents.T he volume component in the 24-h sample and the initial
of stomach oil after we fed chicks 3 ml of a t:t samplew as not significantlyd ifferent).
(w/w) mixture of olive oil and cetyl oleate was The compositiono f stomacho il samplesf rom
5.9 ñ 0.4 ml (n = 6). On the subsequent two chicks on Little Duck in 1986 and 1987 was
nights, chicks received meals from their par- different from that of samplest aken from Kent
ents. The ratio of triglyceride to wax ester ra- Island chicks( Fig. 2). Wax estera nd triglyceride
dioactivity remained constantf or the first 24 h content were nearly equivalent. A component
despite a nearly threefold dilution of both with chromatographicp ropertiess imilar to di-
markers( addition of unlabeledl ipid from meals acyl glyceryl ether was detected. Estimated
fed by their parentsd iluted the markers).A t 48 phospholipid content was 0.21 ñ 0.26% (n =
h post-ingestion,w ax esters apparently were 12). As with the Kent Island chicks, the relative
retained in stomach oils at higher levels than proportions of each lipid component did not
triglyceridesH. owever, < 0.4%o f either labeled changes ignificantly after 24 h in unfed chicks.
lipid was present, and the significanceo f this Thus, the composition of stomach oil is stable
finding is questionableS. ignificantlyf asterg as- for at least 24 h provided no dietary lipids are
tric emptying was observed in chicks fed by eaten. We believe that lipid secretion by the
their parentst han in thosef ed artificially. We proventriculus, if present, is extremely low.
attribute this to larger and fresher natural meals. The compositiono f stomacho il changedw hen
Stomacho il composition.--Ins tomacho il sam- chicksw ere fed nightly meals( Fig. 3: A and B).
piest aken from randomlyc hosenc hickso n Kent After an initial meal of homogenized krill sup-
Island in 1986 (Table 2), the oil contained most- plemented with a t:l mixture of triglyceride
ly triglycerides, with only a small proportion and wax ester (and containing labeled triglyc-
of wax esters (Fig. 2). Cholesterol concentra- eride and triether), we fed subsequentm ealso f
tions,d etermined by TLC-FID (0.12 ñ 0.008 mM, homogenizedk rill supplementedw ith triglyc-
n = 14) and enzymatically (0.14 ñ 0.01 mM, n eride. Each meal (53 kJ) contained ca. 1.0 g of
= 14),w ere statisticallyin distinguishabl(ep aired lipid. With each meal, triglyceride content of
t-test,t = 0.575,d f = 13, P -- 0.575).T riglyceride stomacho il increased (3.68 ñ 0.93%/day, R2 =
concentrationsd etermined by TLC-FID (0.94 ñ 0.246) while wax ester content decreased( 8.36
0.03 M, n = t0) were significantlyh igher (paired ñ 2.52%/day, R2 = 0.319). The free fatty acid
t-test, t = 3.96, df = t0, P = 0.0033) than those content remained constant while the cholester-
Octobe1r 989] StomacOhi l Formation 693
8O
ß Kent Island
LittleD uck
6O
40'
20'
Fig. 2. Stomacho il compositionf rom Leach'sS torm-Petrelc hicks on Little Duck Island (1986 and 1987)
and Kent Island (1986).
ol content increased (6.1 _+ 1.69%/day, R2 = in the field for Leach's Storm-Petrelsp rior to
0.267).T hesec hangesi n lipid compositiona re fledging (Ricklefs et al. 1980a, b, 1985).
consistentw ith replacemento f the volume sam- Additional evidencef or volume regulation of
pled (0.364 _+0 .014 ml, n = 30) by dietary tri- stomacho il was observedin daily weight gains
glycerJde( assumingc onstantv olume). The ini- exhibited by 7 chicks( 70 dayso ld) after removal
tial volume of stomach oil was 7.45 _+ 0.459 ml of all stomacho il. Eachc hickw as weighed prior
(n = 6). Sampling removed 4.9% of the stomach to being fed nightly meals of krill supple-
oil per day. From fecal collections on the first mented with triglylceride (2.2 g/meal). Aver-
two days, 2.3 + 0.53% (n = 12) of [3H]-GTE was ageb ody massi ncreasedl inearly for 7 days( 1.12
excretedw ith chicksa ssimilating9 4.2 _+0 .58% + 0.06g /day, R2 = 0.97)u ntil eachc hickg ained
(n = 12) of labeled triglyceride. The daily de- on average 8 g. Subsequentw eighings indicat-
crease( 7.35 + 0.85%/day,R 2 = 0.705) in specific ed no additional changeJ n mass.A spiration of
activity of [3H]-GTE Jsc onsistentw ith addition the proventriculus on day 9 provided 7.75 _+
of dietary lipid equivalent to the volume re- 1.46g of lipjd, and no net changei n body mass
moved Jn sampling plus the volume emptied from the initial weighing at the start of the
from the proventriculusf or assimilationb y the experiment.
chick (Fig. 3C). This storageo f gastricl Jpid oc- Fateo f polarl ipidsa nd hydrocarbonins s tomach
cursa s body weight decreases(F ig. 3D). Except oils.--Phospholipidsr apidly repartJtionedo ut
for one individual, all chicks had completed of stomacho ils (Fig. 4: A and B). In contrast,
growth.T he dailyw eight loss( 1.81 + 0.21g /day, fatty acidsa nd alkaness lowly repartitJonedo ut
R 2 = 0.93) observed is similar to that observed of stomacho ils (Fig. 4: C and D).
694 PLACEET A L. [Auk,V ol. 106
DISCUSSION
In Leach's Storm-Petrel, we found that stom-
ach oils originate in the diet, but their com-
position need not match that of the diet pre-
cisely.W e determinedt hat extensived ifferential
lipolysiso f wax estersv s. triglyceridesi n the
2 proventriculus was not responsible for the
prevalenceo f wax estersi n the stomacho ils of
[]
some species,a nd that the proventriculus se-
cretesl ittle, if any, neutral lipid. The lipid com-
Days
positiono f stomacho ils reflectsn ot only the
lipid composition of prior meals but also the
relative solubility eachc lasso f lipids hasi n the
stomacho ils previously accumulated.F inally,
1.0 duringt he week or two prior to fledging,c hicks
S o
regulatet he amount of stomacho il by restrict-
0.8
ing the rate of gastrice mptying.
0.6 Physiologicabla siso f stomacho il formation.--
Gastrice mptying in animals is a highly regu-
0.4 lated and coordinatedp rocess.G astrice mpty-
' ing is slow when the stomachc ontainsn utrient-
0.2-
rich food (sucha s lipids) and is more rapid when
the contentsa re less energy rich. In humans,
0 O.C
the half-time for gastric emptying of a non-
Days nutrient saline solution is only 7.9 + 1 min
U. while that for a homogenousm eal is on the
order of 60 min (Smith et al. 1984). In other
mammals,g astrice mptying of lipids is nearly
half that for aqueous components (Jian et al.
1982, Meyer et al. 1986). Comparabled iffer-
ß c
encese xist between passager ates of lipids and
0.18
aqueousc omponents in seabirds (Roby et al.
1989). In Leach's Storm-Petrel chicks, the half-
time for gastrice mptyingo f neutral lipids is 70
h while that for aqueouss olutionsi s only 0.35
h. A unique gastrointestinala natomy and cor-
0.10 respondingg astrointestinaml otility are impor-
,(cid:127)_ 0.08 tant determinants of this slower gastric emp-
o
tying of dietary lipid (Duke et al. 1989,R oby et
Days
al. 1989).W e have describedp reviouslyt he gas-
trointestinala natomya nd motility pattern fol-
lowing a meal in Leach'sS torm-Petrels( Duke
et al. 1989). Low gastric motility, slow gastric
80-
D emptying, and the positiono f the pylorus rel-
ative to the proventriculus all contribute to
stomach oil accumulation.
Fig.3 . Changesin the contento f stomacho ilsf rom
62-day-oldc hicksf ed nightly: (A) triglyceride( .) and
wax ester ([(cid:127)); (B) fatty acid (¸) and cholesterol( 0).
(C) The glycerol triether marker (ß) was diluted by
dietaryl ipids.( D) Prefledgingw eight losso ccurse ven
Days with nightly feedings.
Octobe1r9 89] StomacOhil F ormation 695
,0.8 The mammalian stomach is the site of differ-
ential emptying of lipids and aqueousc ompo-
nents, and it also plays an important role in
-0.6 .(cid:127),
neutral lipid emulsification.I ngestedf ats must
be emulsifiedo r solubilizedi n the stomachp rior
'0.4 0 *(cid:127)
to the formation of micelies in the aqueousc on-
tents of the duodenum. Potential emulsifiers
- 0.2 i(cid:127)) that can function in the acid milieu of the stom-
ach include peptic digestso f dietary protein,
0.0 complexp olysaccharidesa,n d dietaryp hospho-
4
lipids (Carey et al. 1983). Some enzymatic hy-
Time(hours)
drolysiso f triglyceridesis known to occurw hich
resultsi n digestiono f up to 30%o f dietary fats
(Careye t al. 1983).T he monoglyceridesfo rmed
during this gastricl ipolysisa id in the emulsi-
fication process.
In seabirds,w e found very little gastric li-
polysis( Roby et al. 1986,P lacea nd Roby 1986).
'0.5 A possiblee xplanation of the efficient assimi-
lation of nonpolarl ipids is the unique character
of the "enterogastricr eflex." In fowl, asi n mam-
-0 .3 mals,g astricm otility is inhibited by intraduo-
denal injections of 0.1 N HCL, 1600 mOsm so-
lutions of NaC1, corn oil, amino acids, or by
0.(cid:127) intraduodenal balloon inflation (Duke and
0 I 2 3
Evanson 1972, Duke et al. 1973, Sklan et al. 1978).
Time(hours)
An aspecto f this feedbackr egulatory mecha-
nismp eculiart o birds,i ncludings eabirdsi,s the
7.0
occurrence of one or more intestinal refluxes
C1 0.6
ß 0.5 (cid:127) during the period of gastricm otility inhibition
6.5'
(Duke et al. 1973, 1989). Intestinal refluxes occur
0.4 (cid:127)
.,_6(cid:127)0.0(cid:127) ' approximatelyth ree timesm oreo ften in Leach's
Storm-Petrels than in fowl (Duke et al. 1989)
and involve the movement of intestinal con-
0.2 (cid:127) (cid:127)
5.5' tents back to the proventriculus. Thus, gastric
o.1 O) emptying is tied closelyt o the receptivenesso f
the duodenum for additional digesta, and the
5.0 0.0
1' 0 2'0 30
0 reflux returns the digesta( both gastrica nd duo-
Time (hours) denal) for further processingi n the gizzard. In
the processd, uodenalp roductsl ike monoglyc-
eridesa nd fatty acidsa re refluxedt o the gizzard
2.5 '1,0 alongw ith biliary (bile salts,p hospholipidsa, nd
.o.8
Fig. 4. Repartitioningo f dietaryp hospholipids(A )
'0.6 L-a-12-palmitoyl-2-oleoyl-[oleoyl-l-14Cp] hosphati-
dylcholinea nd (B) L-a-dioleoyl,[ dioleoyl-l-"C] phos-
'.0.4 phatidylcholine, fatty acids (C) ([1-(cid:127)4C] stearic acid)
and hydrocarbons( D) (n-[I-"C] hexadecane)f rom
stomacho ils. All comparisonsa re made relative to
'0.2
[sH]-GTE.T he ratio of [3HI #Ci/"C #Ci in stomacho ils
is given on the left ordinatew hile the specifica ctivity
0.0
10 20 30 40 50 (#Ci/ml) of "C is given on the right ordinate.T he
arrow indicatest he initial valuesa t the time of inges-
Time (hours) tion.
696 PL^CEE T^ L. [Auk, Vol. 106
triglycerides)a nd pancreaticp roducts( lipases). low their melting temperature,t he solubilityo f
Gastric production of lipid emulsifiers is re- thesec ompoundsc an be estimatedb y the equa-
placedi n birds by productso f normal intestinal tion:
lipolysisw hich are refluxedt o a highly efficient Log(molef ractions olubility)
emulsificationm ill (the gizzard).W e believet hat = --ASt(Tm-- T)
2.303RT' (3)
intestinal reflux is unique to birds and permits
high assimilatione fficienciesfo r suchn onpolar where ASfi s the entropyo f fusion,a nd Tm is
lipids as wax esters( Roby et al. 1986,P lacea nd the melting point (Patton et al. 1984). For ex-
Roby 1986). We also believe that intestinal re- ample, palmitic acid (melting point 61.8øC)h as
flux is responsiblefo r gastrice mptyingo f polar a 0.76% (w/w) solubility at 14øCw hile at 37øC
lipids dissolvedi n stomacho ils. the solubility is 4.72% (w/w) in trioleoylglyc-
Effectso f solubilityo n stomacohi l composition.- erol.
Lewis (1966) found glyceryl ether diesterst o Recently,t he presenceo f fossil fuel hydro-
dominate stomach oils from Leach's Storm-Pe- carbons in stomach oils has been used to mon-
trels. Lewis (1966) felt the presenceo f glyceryl itor water quality in the marine environment
ether diesterss upporteda secretoryo rigin, be- (Boersma1 986). Our findings with n-hexadec-
cause these compounds were not common in ane indicate that ingestedh ydrocarbonsp ersist
zooplankton and other marine organisms.O ur in stomach oils of Leach's Storm-Petrels at least
results establisht hat the composition of stom- as long as the nonmetabolizablel ipid marker
ach oils in Leach's Storm-Petrels is determined (TMRT > 70 h). Becausere siduef rom a single
by dietary lipid and the composition can be contaminatedm eal may persisti n stomacho ils
highly variable( e.g. Fig. 2). Stomacho il com- for extended periods, care must be taken in as-
positioni s further influencedb y the solubility sessingf requencyo f contamination.
of each dietary lipid classi n previously accu- Physiologicaald vantageso f stomacho il forma-
mulated stomacho ils. When major dietary lip- tion.--An energetica dvantagec an be realized
ids are changedt, he compositiono f the resident by metabolizings tomacho ils in preferencet o
stomacho il changesb ut not in a manner nec- fat from adiposet issued uring fasts( Roby et al.
essarilyr eflecting the lipid compositiono f a 1989). Such gastrics torageo f lipids precludes
meal( e.g.F ig. 3). The absenceo r low abundance the energy costso f synthesizingt riglycerides
of polar lipids (such as fatty acids and phos- from assimilatedf atty acids, transporting the
pholipidsi n stomacho ils) is due to limited sol- triglyceridest o the fat depots,a nd of later mo-
ubility in stomacho ils and rapid gastrice mp- bilizing those energy reserves.T hese energy
tying of these polar lipids with the aqueous costs amount to ca. 25-30% of the assimilated
phase( Fig. 4). energy (Ricklefs 1974,S padye t al. 1976).
Lipid analysiso f mealsd eliveredt o chickso n A correlateo f increasingt he energy density
Kent Island recorded nearly 21.7 ___1 0.5% SD of a meal through concentrationo f dietary lip-
phospholipid (Place and Roby 1986) yet the ids is a lower dietary osmotic( i.e. salt) load for
phospholipidc ontent of stomacho il was neg- the chick. Marine prey items (particularly in-
ligible (<1%). We can explain this from the vertebrates)i mpose large salt loads. Excessd i-
physicalp ropertieso f phospholipids.P hospho- etarys altsa re excretedf rom the nasals altg lands
lipids are insoluble in neutral lipids (Sander- through energy dependentm echanismsw hich
mann 1979)a nd partition into the aqueousp hase might consumer eserveso therwisea vailablef or
either as monomerso r as micelies.P artitioning maintenancea nd growth. Leach'sS torm-Petrels
enriches neutral lipids as the aqueousp hase of produce highly concentratedn asal secretions
a meal emptiesr apidly from the stomach.T he (Schmidt-Nielsen1 960). Through inclusion of
solubilitieso f other polar and nonpolar lipids stomach oils in chick meals, procellariiform
in stomacho ils (sucha s long chain fatty acids, chicksm ay experiencel ower costso f digesting
alcoholsa, lkanest, riglycerides,a nd chlorinated marine prey items.
aromatic and aliphatic hydrocarbons)a re de- Salt load reduction also lowers a meal's cal-
terminedl argelyb y their meltingp oints.A bove cium load, which for marine prey items can be
their melting temperature( i.e. asl iquids),a ll of high. In fat absorption,h igh dietary calcium
thesec ompoundsa re theoreticallym isciblew ith levels frequently result in calcium soap for-
liquid trioleoylglycerol( Pattone t al. 1984).B e- mation of fatty acids and subsequentp oor ab-