T}Ji: ir?TAKE Ch' rdEE rATIY aCIDC VHJ''' '>:a WATER fv
A KATilNE FILTER FEEDER, CiVl.^^airirTtvi/l VIUGWICa"
TERKf AUEN BUNDE
A U.iSSEETATION rEESEWTED TO THE GRADUAT/^ COUNOJL C^^
THE mNTJVF.RSIT"! OF .^XOBIDA
xN -AI^TIAL FiTL-imiENT 0? THE RLQUIREHENIS FOR T.iE
DEGR;:;f; CF DOCTOR OF PHILO.S'OFK.Y
miVERE'T^- OF FFORIDA
1975
ACK.NOTsTLEDGEJ'TEKTS
The author wishes to express his sincere appreciation and gratitude
to his research director. Professor Melvin Fried, for his guidance,
encouragement, and generous support during the completion of this work.
The author also wishes to express his appreciation to the members
of his supervisory committee. Dr. Charles Allen, Dr. William Carr , and
Dr. Samuel Gurin, for their suggestions and criticisms during the execu-
tion of this research.
Special tlianks are given to the author's fellow graduate students
for the suggestions and encouragement they offered. Thanks are alt^o
offcr.-fl espec:?ally to Kr. William Gilbert for hlij assistance in the
preparation of t!ie computer programs.
A very special thanks is also expressed by the author to his
parents, who have made his education possible, and to his wife without
whose understanding, patience, constant encouragement, and Jong hours
of typing, this work would never have been completed.
TABLE CF CONTENTS
ACKNOWLEDGEMENTS
LIST OF TABLES
LIST OF FIGURES
ABSTRACT
INTRODUCTION
Metabolic Significance of Dissolved Organic Matter
Lipids and Free Fatty Acids in the Marine Food Chain'
r.ie Oyster as a Possible Experiinental Subject for Lipid
Uptake _
Research Objectives ....
WiTERIATS A^JD METHODS .
DATA AllD DISCUSSION
CONCLUSIONS . . . ,
BIBLIOGEAPm;' . . , .
^>lOG.liA]']\JCAL SKETCH
Page
ii
iv
V
viii
1
11
14
16
17
Materials
Methods ... * -'-''
' = . . - 20
31
Lipjds and Frds Fatty Acids in Sea Water ... -.i
Uptake of Palmitic Acid :,;:
Ceiltc Uptake Experiments '
CoTicentration Dependent Uptake--Kinetic-Parameters *of '
Uptake . ,
Lipids of Craososf.r'sa and the Incorporation of'LabeJed'
fatty Acids
Competitive Uptake .'.'.* J7-,
Turncver of Lipid Classes .....'''''
50
60
97
105
110
116
120
xai
LIST OF TABLES
T^bl^ ^ Page
1 Amino Acids and Glucose Uptake . . . . , y
2 Fatty Acias in Marine Waters 3 2
3 Visualization Reagents for TLC 27
4 Concentrations of Excractable Specific Lipids in the Sea
Water Collected on June 21, 1974 (Extract A) and
1-larch 31, 1975 (Extract B) 3/,
5 The Free Fatty Acids In the June 21 Sea Water
Extraction
38
b Localization of Oil Red 0 Celite ParticJes Removed fron^
Sea Water by Experimental Animals . 5/j
7 The Effect of Oleic Acid on Steoric Acid Uptake 101
8 The Effect of Oleic Acid on Palmitic Acid uptake 103
9 The Effect of Palmitic Acid on Oleic Acid Uptake .... 104
XV
LIST OF FIGURES
Page
The Flow of Organic Compounds in the Marine Ecosystem . . 3
Cycling of Organic Matter by the Benthos 4
Extraction with Adapted Bloor Method 21
4 Extraction with Adapted Bligh and Dyer Method 22
Separation of Polar Lipids in Sea Water Extracts
6 Separation of Neutral Lipids in Sea V.'ater Ext
Figure
1
2
3
5
7
8
10
16
18
o
o
racts . . . 33
Gas Liquid Chromatograph of Fatty Acid Methyl Esters
Prepared from Sea Water Extract of June 21 37
Diffusion of Adsorbed Labeled Fatty Acid into a Sna Water
Wash Saturated with Unlabeled Palmitate 40
The Uptake of Palmitic Acid Measured Usin? the Bloor
Extraction Technique ....
•■•-•• 43
Removal of ^''c Fatty Acid by Background Adsorptioa onto
Shells and Glass Surfaces 4^
11 The Uptake of Palmitic Acid in the Presence of 200 mM
Sodium Cyanide .
47
12 The Uptake of Palmitate, Doub.le Addition of Label .... 49
13 The Uptake of Palmitic Acid by Open-Shell Animals .... 51
14 Temperature Dependent Uptake cf Palmitate r,,
15 The Uptake of Celite-adsorbed Palmitate
55
The Uptake of Celite-adsorbed Palmitate, Open Shell
Animals . . .
' 57
17 The Uptake of 2.8 x lO'^ M Paimi.ate, Celite-adsorbed
and Free . .
59
The Uptake of 2.8 x lO"^ M Stearate, Celi te-.adsorb.d
and Free . . .
62
LIST OF FIGURES— Continued
Figure p^gg
19 Concentration Dependent Uptake of Palmitate 64
20 Concentration Dependent Uptake of Stearate 66
21 The Concentration Dependent Rate of Uptake of
Palmitate , 67
22 The Concentration Dependent Rate of Uptake of
Stearate 63
23 Lineweaver-Burk Transformation of Palmitate Uptake
Data
24 Lineweaver-Burk Transformation of Stearate Uptake
Data
70
71
25 Concentration Dependent Uptake of Oleate 74
26 The Concentration Dependent Rate of Uptake of Oleate . 76
27 The Thin Layer Chromatographic Separation of Oyster
Neutral Lipids yfi,
28 The Thin Layer Chromatographic SeparaLicu of Oyster
Polar Lipids ........... 79
29 Radiochromatographic Scan of the Neutral Lipid TLC
Separation , 31^
30 Radiochromatographic Scan of the Polar Lipid TLC
Separation 83
31 The Two-dimensional TLC Separation of Oyster
Phospholipids 85
32 Gas Liquid Chromatograph of Fatty Acid Methyl Esters
Prepared from Esterified Fatty Acids of Isolated
Oystar Triglycerides 87
33 Gas Liq.iid Chromatrgraph of Fatty Acid Methyl Esters
Prepared iron Esterified Fatty Acids of Oyster
Total Lipid Exr.racts .' 89
34 Incorporanion of "'^C labeled Palmitate into Isolated
Lipid Classes 9]
35 Concentration Dependent Inrorpurat-ioi: of Palmitate into
Phosphatidyl Choliiie . , ,. 94
VI
Figure
37
38
39
42
LIST OF FIGURES— Continued
The Concentre, tiou Dependent Rate of IncorDoration of
Palmitate into Phosphatidyl Choline
Lineweaver-Burk Transformation of Palmitate Incorpora-
tion Data
Concentration Dependent Incorporation of Stearate
into Total Phospholipids
The Concentration Dependent Rate of Incorporation of
Stearate into Total Phospholipid
40 Lineweaver-Burk TransformaLion of Stearate Incorpora-
tion Data
41 The Turnover of Lipid and Non-lipid Compounds Labeled
with [-^HjAcetate ...
The Turnover of Specific Lipid Classes in the
Chloroform Extracts of Oysters Labeled with
[■^HJAcetate
Page
95
96
98
99
100
106
109
Vil
Abstract of Dissertation Presented to the Graduate Council of the
University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy
THE UPTAKE OF FREE FATTY ACIDS FROM SEA WATER BY
A MARINE FILTER FEEDER, CRASSOSTREA VIRGIIUCA
By
Terry Allan Bunde
June, 1975
Chairman: Melvin Fried
Major Department: Biochemistry
The ability of the American oyster, Crassostrea vir-ginica^ to
remove naturally occurring dissolved free fatty acids, j.n concentrations
approximating those found in sea water, vas iavestigaucd using radioactive
isotopes of palmitate, stearatc, and olcate.
Petroleum ether (30 - 60°C) extracts of the sea water from a Flor^.da
Gulf Coast estuary contained up to 280 yg of total lipid material per
liter including 77 yg of free fatty acid. Th^: fatty acids, separated by
gas liquid chromatography, were predominatclj saturated with even caibor
numbers. The major fatty acid pj-t:sent was palmitate.
The animals were shown to remove labeled palmitate from sea water by
measuring the appearance of the radio-activity iu the chloroform extract-
able material. The uptrke process was shov:n to be physiolcglcsl erd rot
chemical adsorption onr.o shells. This assimilation was Inhibited ,7±za
200 ml-I sodium cyanide. The te.-peratcre dependence of the uptake process
vas investigated at 20, 25, 30, and 35^C.
The rate of uptake of 50 pm celite particles carrying adsorbed
radioactively labeled stearate r,nd palmitate dejaonstratcd that the process
VJ.1.1.
of filtration feeding was not responsible for the removal of freely
dissolved fatty acid.. The rate of uptake of celite bound material was
delayed by 3C minutes when compared to the uptake of an equal concentra-
tion of dissolved acaterial.
The kinetics of the uptake into chloroform extractable material .^as
investigated for palnitate, stearate, and oleate. Both palpitate and
stearate shoved saturable uptake systems as detemined from reciprocal
rate-concentration plots. The rate of uptake of both acids markedly
increased when micellar concentrations of the fatty acids were reached.
The rate of uptake of oleate was ,nuch less than that for palmitate and
stearate, and was not saturable at natural concentrations.
Ihe rate of uptake into isolatable lipid classes was investigated;
the major species labeled were phosphatidyl choline, triglycerides, and
cholesterol. The rates of incorporatron of palm.t.te i.to phosphatidyl
choline and stearate into the total polar lipids were determined.
Oleate was shown to effectively inhibit the uptake of stearate in
competition experiments, but no effect was seen by oleate on the palmitate
uptake. Increased oleate concentrations were shown to promote palmitate
•uptake.
■ Turnover^rates for various lipid classes were determined by labeling
vith sodiu. [^'acetate, removing the label, and following the decrease
in specific activity of each lipid with time.
The contribution of th. uptake process to the total metabolic needs
of the animal was estimated. The impact of such lipid uptake studies was
discussed in Irgh;: of municipal sewage and petrochemical pollution of
naturaj ovs^pr VisN-; ^-it-., ^ i -.
. ^}s.er ha.,. ..at.- a.. weU as the selection of oysters as a possible
squaciilr;ure sp-,^cies.
3X
INTRODUCTION
Metabolic Signif jcance of Disgolved Orf^anlc Ma 1 1 er
The salt waters of the world contain relatively constant concentra-
tions of inorganic compounds, evidencing only small changes in salinity,
but they show orders of magnitude variation v/ith tlir.e and location in
concentrations of dissolved organic matter and diasoived particulate
matter (Wagner, 1969; Duursma, 1961). Early investigations of dissolved
organic substances were hampered by crude ruethods of sampling, analysis,
and quantitation; but with newer, uore refined techniques, it has become
apparent that the oceans of the world contain more dissolved orp?nic
matter than that which is represented by the entire living bioiiass of the
oceans (Duursma, 1961). All major classes of biologically important
organic molecules are found in sea water: amino acids and peptides,
simple and conjugated carbohydrates, nucleic acids, and jiplds. These
materials share the comicon property of being able to pass through a
0.45 ',im cellulose acetate filter and are, therefore, distinguishable from
the particulate matter which such filters retain. The concentrations of
these molecules vary within fairly wide limits from one body of water to
another depending upon the season, the metabolic activity of the ecoj;} stem,
the depth of the water, and the specific flora and fauna found in the
water,.
The methods by which these compounds have been analyzed involve
techniques such as dialysis, adsorption, ion-e>:change, solvent-extraction.
a^v2 co-preclpitation (Wagner, 1969). The difficulties inherent in B,easuring
mg/llter or yg/liter quantities of organic con^pounds in solutions containing
g/liter quantities of Inorganic salts have made quantitation difficult, but
reliable data show cotal amino acid concentrations of 30 yg/liter of vrhich
.16 pg/liter is glycine (Hobble et al. , 1968); carbohydrate concentrations of
0.5 mg/liter (Okaichi. 1967); and lipids in 1-10 mg/liter quantities
(Jeffrey, 1966).
The sources, and energy and matter InterreJationships of this huge
reservoir of organic matter are not specifically known, but several possible
pathways have been investigated. The best description is derived from a
figure in a review by Duurs.n. (1961) which is Figure 1. This flow diagram
depicts the dynamic nature of the pool of dissolved organic solutes and its
relationships to the several pathways of decomposition, excretion, and leak-
age Which r.sult in these molecules. The primary producers in a salt water
ecosystem, the phytoplankt^r.^ have boen shown .o lose a large amount of
their photosynthetic products through leakage and overproduction, up to
1-40 mg Carbon/m~' sea water/day depending upon the water depth and latitude
(Thomas. 1971). The zooplankters which consume the primary produce.rs, also
-leak organic molecules into the pools of both dissolved and particulate sub-
stances (Johannes and Webb, 1965). This complex relationship between the
various organisms and the organic matter, and the probable importance of
bacteria in processing dissolved organic matter, are outlined in a figure
derived from Johnson (1974) which is Figure 2. The physical and environ-
mental forces involved in the production and processing of organic .atter by
the benthxc animal communities are as complex and as important as the Lio-
chemical interconversicns that occur. The pools of detritus and dissolved
organic matter a.e not st.tic but in a constant dynamic state as ere the
organisms at eacii trophic level.
Light
v.yL
Living Organic
) Matter
" 4-
ApsimiJaLion
i.
Excretion
I Filter \ > Dying and
\ Feeding \ V,xcr<^.tion
\ Assimilation
of Dissolved
Organic Matter
\
Exc
Overp
Excretion and \
verproduction \
\ ^ Decomposition
Rain
Atmosphere
V
TJ.
NO3-
»2
NO,"
II2CO3
-."
HCO,"'
NH3
_0
C03^
Mineralization
Sedimentation
Dissolved Organic
Carbon, Nitrogen,
and Phosphorus
Bacterial
Inter conversions
A
\j/ Precipitation
iJTTmiTf
Fresh Water Runoff
Figure 1. The Flov.' of Organic Compounds in rhe Marine Ecosysteia,
Taken from: Duursnia, E.K. Netherlands J. of Sea Pes. 1:4
,(1961).
Macroscopic Plants
and Assorted
Epiblota
AUTOCHTHOKOUS
SOURCES
Dissol^'ed
Organic *:
Matter
ALLOCHTHONOUS SOURCES
Phytoplaiikton,
Zooplankton, and
Their Feces
Organic
Precipitates
I
Terrestrial
Organic Matter
BENTHOS
/
I
,1
Microflora and
Protozoans
BACTERIA
Respiration
Feces and Pseudofeces
Floe Aggregates
Encrusted Mineral Grains
^ Export
Accumulation
in Sediments
Figure 2.
Cycling of Organic Matter by the Benthos.
op't^fT'n '"^'" ^°" ''°^"^°" ^^^7^) ^^Pi-ts the function
of or-L ' °' '°"°" '""'"'"S organisms in the processing
of organic matter. The external sources of organic matter
aie shown at the top and the sinks at the bottom l^e
^:j::Ci ;:;to^?:e:irdi:-?e^najt:r"" ^^ -°— ^ -^
Taken from: Johnson, R.G. J.Mav.Res. 32(2) :326 (1974) .
In 1908 a German biologisL, PGtter, concluded, from the crude
analytical data on the concentrations of dissolved organic matter in sea
water which were available to him, that this pool of organic molecules
was a valuable and even necessary resource in the nutrition of marine
organisms (Patter, 1908). His theory was considered valid until Krogh
(1931) slio^ed that Patter's determinations of the concentrations of those
materials erred significantly on the high side, and held that, although
there were amino acids, carbohydrates, and lipids in sea water, they were
not present in sufficient quantities to be a valuable energy source. In
a later paper (as reviewed in Duursma, 1961) Krogh acknowledged that some
organisms could remove these molecules but still held that they were not
energetically significant. The current concepts of what is indeed meta-
bolically significant to an organism were formulated in a series of papers
by Lucas (19A6, 1949. and 1961), whose views of utilisation involve the
idea of pools of molecules providing necessary metabolic intermediates
and "essential molecules" for the organism, rather than functioning as
significant sources of nitrogen, carbon, and phosphorous for metabolic
energy. However, if an organism did possess pathways for assimilation of
these molecules for anabolic or catabolic needs, then such pools of
organic molecules in the sea could be very important.
Kith the presence of dissolved organic matter in sea water an
uxxdisputed fact, research was initiated into elucidating the physical and
chenical forces that convert these organic compounds into particulate
matter of sufficient size for filtration cr adsorption methods to be
used by marine organisms in their removal from the sea water. The initial
studies employed fish hemoglobin as a substrate for coaxescence of organic
matter from sea water. This .aaterial was then shc-vm to be important in
ths nutrition of lnvcrt<ibrates In feeding experin,e„ts CKox et at., 1953),
St.die. into the geophysical forces involved ,„ those sea surface currents
tnown as Langcuir circulation and the produetlo:, of foam lines at thelr
Interface led researchers to formulate the thesis that organic partic.-
lates formed at disturbed air-water interfaces (Baylor et al. , 1962,
Hiley, 1963; Sutcliffe .. al. . 196,,. The formulation of organo-pK.sphate
containing particles at such disturbed surfaces, and the resultant eccumu-
l.>tlo„ of phosphate containing material when these particles were iso'sted
and added to 0.« „m filtered sea water are examples of the process
thereby these particles may be fo.,»d and increase in si.e (Sutcliffe et al
1963)
Baylo. and Sutcliffe isolated organic particulate .atter fro.
despo.ated sea water following filtration throu,. a 0.45 u. filter and
deinonstrate^' '-'-q sn-^TTiroi r.f /^j.
su.M-.al of Avtama cultures fed this mar=.-<^i jo^-
ll-iu days (Eaylor et al. 1963^ t\^ ^.,i.
.-, .1963). Ihe cultures survived and grew as well
.. those fed yeast e.^tracts. The data of these investigators seemed to
.-pport patter's orlglnel ideas and provided an impetus for further work
..a.^ed on the hypothesis that dissolved organics were metaboUcalJy impor-
tant to at least some Invertebrates.
. The work of Fox. Baylor, Riley, and Sutcliffe indicated that
particulate generation was required for feeding. They .said nothing about
-reas in which no significant physical condensation of organic molecules
could occur, but where freely dissolved molecules existed. Grover C.
Stephens and co-workers showed in work published from 1961-1973 that
dissolved free amino aelds and carbohydrates, at naturally occurring con-
centrations, were removed from sea water solutions by several marine
species (.,ee Table 1). Eased on results with radioactive tracers, the
Table 1. Amino Acids and Glucose Uptake.
Author
Collier
et al.
Stephens it
Schinske
Stephens &
Schinske
Reish &
Stephens
Anderson &
Stephens
Taylor
Chiea
e t O.L.
Shick
Date
Organic
Compound
1953 Glucose
1958 Amino Acid
(Glycine)
1961 /jnino Acid
(Glycine)
Stephens 1962 Glvcose
Stephens 1963 Amino Acids
(Ala, Gly)
Stephens 1964 Amino Acid
(Glycine)
Stephens ^ 196f^ Amino Acids
Vi rkan
Organicra
1969 Amino Acid
(Glycine)
1969 Ajnino Acid
(Glycine)
1969 Glucose and
Amino Acids
Crassostvea
virginica
12 invertebrate
phyla
11 invertebrate
phyla
Fioigia
scutaria
Clynenella
torquata
Nereis
limnicola
and sucoinea
Ophiaotis
arenosa
Neantkes
arenaacodentava
Crustaceans
Nei'eis
jire'iis
and saPS
1972 Amino Acids Glyoere
1973 Amino Acid Auveli-a
(Glycine) aicrita
Concentration
Tested
1 X 10
-3
2 X 10
"3
-3
2 X 10
A. 7 X lO'^
~6
1 X 10
2 X lO"^
2 y. 10
1 X 10
-8
-■7
-7
1 X 10
4.3 X 10
2 X 10~^
-7
-6
1 X 10
1.27 X 10
dissolved organic matter could partially meet the energetic needs of
these organisms. By the use of radioactive tracer techniques Stephens
has surveyed several invertebrate phyla: coclenteraces, annelids,
crustaceans, m.olluscs, and echinoderms, and showed that at least some
capacity to remove dissolved free amino acids or carbohydrates exists in
a]l of them. In these studies the disappearance of a tracer molecule
from sea water was monitored as was the appearance of label in the whole
animal digest or extract. The rate at which this process proceeded at
naturally occurring concentrations was used to determine the maximum
amount of assimilation into the organism with time. Knowing the meta-
bolic oxygen consumption of an experimental animal, the percentage of
total carbon influx that was represented by dissolved organic solutes
vras determined.
Perguscn, using an autoradiographic technique to study the uptake of
r.rdno acids by starfish, has shown that these animals could remove the
label from sea water and that the amino acids, first localized at the
surface of the animal around the p.eudopods, were later transported
throughout the water vascular system. The uptake was quantitated by
counting silver grains in the photographic emnlsious (Ferguson, 1970,
.1971). Other work with starfish species (Pequignat, 19:^2) demonstrated
amino acid uptake into an isolated .rm of Henricia c .nquinolenta by
autoradiographic techniques. The labeJed araino acid, as rn Ferguson's
studies, could be seen to b. incorporated almost exclusively into the
ambulacra and aboral w.ll of the arm pJaced in the sea water. Time
course studies revealed major incorporation of the amino acid into
proteins of the gonadal tissue. Indicating significant .trlization of
this dissolved material assimxlatea from sea water.
It is apparent from these studies that animals v;ith soft body tissue
surfaces exposed to the sea water can remove and assimilate dissolved
material in a manner different from their normal feeding habits. Polychaetes
are detritus feeders, bivalve molluscs aru f i] ter-feeders , starfish and
urchins are herbivores, and coelenterates are carnivores, but all appear to
have pathways for direct assimilation.
With the discovery and biological characterization of the pogonophorans ,
much attention was given to the possible mechanism of nutrition in these
benthic invertebrates which possess no digestive system (Little and Gupta,
1968, 1969; Southward and Southward, 1970, 1971, 1972). Amino £cid uptake
from ambient sea water concentrations of lO"^ - 10 "^ M was shovm , followed
by autoradiographic studies of its localization (Little and Gupta, 1968;
Southward and Southward, 1968). Further work yielded data concerning the
uptake of several different amino acids, hydrolysates of algal proteins
(peptides), glucose, and fatty acids (Little and Gupta, 1968; Southward and
Southward, 1970, 2971, 1972), Uptake of such compounds by the pogonophorans
apparently differs only from that in the polychaetes (Taylor, 1969; Stephens,
3 964) in that the efficiency with which pogonophorans remove dissolved
substances is much better, i.e., they are better adapted to environments
wherein con.:-n trations of amino acids and fatty acids are less than lo""" M
(Southward and Southward, 1971). The studies of these animals indicate that
as much as 50 percent of their metabolic needs can be net by the dissolved
organics in the sea water around then;. In pogonophorans, therefore, the
ability to remove dissolved molecules is not accessory but is necessary for
their basic nutrition. They have developed mechanisms that are finely
tuned to ambient organic concentrations so that optir.ium usage of such pools
can be maintained.
10
The basic in vivo experin^ental techniques of Stephens and indeed
of all ethers who have looked at uptake of dissolved material from sea
water, i.e., the use of tracer i^ethods to yield son-.e indication of the
percentage of the metabolic needs met by these substances, have been
challenged by Johannes et al. (1969), In experiments ^ith the marine
turbellarian Bdelloura, these workers found that this animal leaked amino
acids into the medium at a faster rate than it re^noved them from solution^
therefore, any discussion of uptake satisfying net metabolic needs is
incorrect. However, Stephens, in a later paper (Chein et al. , 1972),
showed rhat when a section of body wall of the blood worm Glyaei'a was
removed and placed in a Ussing cnamber in which the flux of amino acids
into and out of the organism could be measured, the not flux was into the
animal.
The raetabolic significance of the work with aaino acid uptake is
complicated by the function of the molecules as ocmoregulators in marine
and estuarine invertebrates. Glycine, proline, alanine, aspartic acid
and the sulfonic acid taurine are all involved in osmoregulation (Gilles
and Schoffeniels, 1969). The uptake of these amino acids from sea water
must be considered in the context that any reverse flow out of the
organism functions to maintain osmoregulation. Stephens looked at the
influence of salinity on the uptake of glycine by Clym^ella torq^^ta
and showed that at low NaCl concentrations the uptake was virtually zero.
At these salt concentrations this animal would be actively lowering its
internal pool of amino acids to comrensate for decreased ionic concentra-
tion in the medium.
The ability to remove dissolved amino acids and carbohydrates at
isoionlc sea water concentrations, however, is real and their net movement
11
into the organism may be iraportant for a broad spectrum of organisms in
which such pathways are not the main nutritional mechanism.
Lipids and Free Fatty Acids in the Marine Food Chain
The organic molecules which have been most exhaustively examined to
date have been the amino acids and glucose; but there is a large and
equally important class, the lipids, which are present in sea water at
metabolically significant concentrations. Table 2 is a compilation of
data from several laboratories on the concentration of lipids, specifically
free, fatty acids. The variability of the data comes from the diverse
methodologies used in sampling, storing, filtering, and extracting the
specimens as well as to differences in source. The latest papers use
filterability through a 0.45 ym filter to define dissolved matter and
employ solvent extraction to separate the lipids.
It is certain that there are large amounts of hydrophobic lipoidal
material dissolved in the oceans of the world, not just in isolated areas
of phytoplankton slicks or polluted coastal waters. While Stephens and
many others were conducting investigations on dissolved amino acids and
carbohydrates, only two investigators were working on the uptake of
dissolved free fatty acids. Southward and South^-'ard (1971, 1972)
described experiments with pogonophoran species, and Testerman (1972)
published data on two nereid species. These experiments demonstrated
uptake processes for fatty acids that were saturable and inhibirable by
other fatty acids. Such uptake operated efficiently at the free fatty
acid concentrations to which the organisms arc exposed in their natural
environment. The fatty acids, once rer.oved, were incorporated into
several complex lipid compounds. The loss of label from these organisms
12
rable 2. FaCty Acids in Marine Waters,
Compound
Fatty Acids
Fatty Acids
Method of
Extraction
Concentration
Liquid-Liquid//
pH 3//ethyl acetate
Liquid-Liquid//
CCl, + CHCl,,
4 J
in mg/1
0.1 - 0.
0.01 - 0.12
Investigator (s)
Slowey
et al. ,
1959, 1962
Williams ,
1961, 1965
Lipids
Liquid-Liquid//
pH 2/ /petroleum
ether + ethyl
acetate
0.4 - 8.0
•Jeffrey ,
1962, 1966
Fatty Alcohols,
Acids, Esters,
• and HC
Coprecipitat: on w/
FeCl //extract w/
CHCl^
0.2 - 1.0
Garrett;
1967
Fatty Acids
Liquid-Liquid//
pH 2.0-2.5//
Extract w/CHCl
0.01 - 0.025
Stauffer &
Macintyre
1970
Lipids/Fatty
Acids
Liquid-Liquid//
pH 2//CHC1
(saponf ication)
0.11 - 0.13
0.06 - 0.05
Testerman,
1972
Source: Taken in part from Jeffrey (1970) and Testerman (1972^
13
into the medium, the so-called "leakage" rate, was orly 5 percent, with
the majority of the "leaked" radioactivity being in the form of CO^
indicating the catabolism of the free fatty acid (Testennan, 1972).
The work on lipid uptake by marine animals does not suffer from some
of the problems of amino acid experiments. The lipid material, due to
its hydrophobic nature, is not as freely soluble as amino acids. The
lipophilic compounds involved are not readily diffusible in nature and
are not involved in osmoregulation processes as are the amino acids.
After a lipid compound is transported into an experimental animal, the
reverse diffusion rate back into the water is not expected to be as large
as that for amino acids; hence, the major direction of the movement is
into the animal. Therefore, this movement may be much more metabolically
significant.
Increasing coastal pollution problems ascribed to oil spills and
natural oil seepage from the sea floor have caused several laboratories to
investigate the effect of petroleum hydrocarbons on lamellibranch molluscs
(Lee et al.^ 1972; Fossato and Siviero, 1974; Stegeman and Teal, 1973).
These investigations showed that CrKLSSOstrea vivglnica and Mytilus edulis
were able to remove significant quantities of sub-lethal concentrations of
petro-lipid material, up to 50 yg/gram wet body weight. This lipid material
was assimilated in the gill and mant J e areas as well as in the gut, indicating
a possible direct adsorption paLhv;ay (Lee et at. ^ 1972). The naturally
occurring hydrocarbons in the lipid pocjs of the organisms vrere not as
saturated nor as aroir.auic iu nature as the exogenous pt tro-hydtocarbons
and were not effected by the la::ge concentrations of the foreign compounds.
Stegeman and Teal (1973) found that the fat content of nhe animal was
proportional to the maximum ability to atore the foreign hydrocarbon
14
material. This would seem to indicate that, once removed, the material
mixes with the lipid pools of the organism.
The Oyster as a Possible Experimental
Subject for Lipid Uptake
Studies on the feeding behavior of the American oyster, Crassostrea
vir>ginioa, have been designed to determine the type and approximate size
of particles filtered, and the nature of the filtering process. Because of
the economic importance of the species, much of this work is reported in
Wildlife Fisheries bulletins and other governmental publications, and deals
with growth rates almost exclusively (Collier et al. , 1953; Galstofi, 1964;
Korringa^ 1952). The work that has been done concerns the filtration system
of oysters and its ability to remove the several size classes of organic
material which make up its diet (Haven and Morales-Alamo, 1970). The results
indicate that oysters filter several different classes of material:
(1) dissolved organic material 0.8 - 1.5 pm, (2) nano- and ultra-plankton
5.0 ym, (3) marine bacteria 1.0 - 2.5 Mm, and (4) macroparticulate organic
matter 1 - 10 ym and larger. Data from Ward and Aiello (1973) on the mussel
I'lytilus edulis , a lamellibranch like Crassootvea^ imply that the gill is
a uual purpose organ serving both as a surface of oxygen exchange and as an
ultra-structure for ciliary-mucoid filtration. The controversy surrounding
the importance of the mucus strand in entrapment of particles smaller than
the interfilamental ostia of the gill has not been resolved, but it now
appears likely that the structure of the gill lamellae can filter particles
down to 1 ym in size without mucus (Haven and Morales-Alamo, 1970).
The first in vitro work on uptake by Ir.mellibranchs showed that the
gill tissue is the most importsnt site in the animal for free amino acid
and sugar uptake (Bamford c-nd Gingles, 1974; Bamford and McCrea, 1975),
By excising gill cissue frox Cci^astodenna edule^ the common cockle, and
measuring the uptake of C labeled amino acids, these workers demon-
strated that the uptake mechanism is saturable, has a diffusion coioponent,
and that there is inhibition by other amino acids. Their work with the
Japanese oyster, Crassostrea gigas, involved the uptake of labeled glucose
and the inhibition of such uptake by glucose analogs. The impetus for
this work came from a series of autoradiographic studies by Pequignat
(1973) on the uptake of amine acids and glucose by Mytilus edulis. In
these whole animal experiments, labeled amino acids, removed from sea
water concentrated in t.'ssues of the mantle, foot, and gills, i.e., those
soft tissues exposed to crganics in the water as it passed through the
shell. It i? obvious that the gill is vitally important in the feeding
process both for large macrcuolecular aggregates and detritus in filter-
feeding and for direct assimilation of dissolved material.
The metabolic importance of la.r.sllibranch filtration of sea water
can be expressed in the following energetic calculation derived from
Nicol (1970). The oyster can filter sea water at a rate of 3 liters/hr
during which time it consumes 0.20 ml of 0^; this rate of filtration may
then be expressed as 15 liters H^O.'l ml O^. If 1 ml of 0^ will oxidize
0,8 rag of organic matter, and if the basal metabolism represents approxi-
mately one- third of the total ox-ygen consumption, the amount of organic
matter that must be removed from the sea water is
0 8
YJ- x 3 = 0.15 mg/liter.
Nicol suggests that the particulate diet of oysters, detritus and phyto-
plankLon, can provide 0.14 - 2.8 mg of organic matter/liter. Since the
results of in vitro and in vivo uptake experiments with lamellibranch
16
molluscs (BaTiford and Ginglcs, 1974; BaiTiTord and McCrea, 1975; Pequignat,
197.3; Stephens, 1963), show that dissolved material, present in concentrations
up to 10 mg/nil, can be removed from sea water, dissolved organic matter shoalc
be considered as a possible source of metabolic energ)'^.
Research Objectives
The purpose of this research was to study the uptake ar.d incorporation
of dissolved free fatty acids by a marine filter-feeding mollusc, the
American oyster, Ci-assostrea virginica. To formulate and organize the
objectives, the following questions were asked:
(1) What are the ambient concentrations of free fatty acids in the
water in the Cedar Key estuary and w^hat is the free fatty
acid distribution?
(2) Can the oyster remove free fatty acids from sea water at those
concentrations found naturally and are the free fatty acids,
once removed, incorporated into the lipid pools cf the
organisms?
(3) Is this uptake a saturable process? If so, what are the initial
rates and concentration dependence of the process or processes?
(4) How dees the uptake of dissolved material (i.e., smaller than
0.45 ym in diameter) compare with the uptake of particulate
material 50 lim in size?
(5) Is there any temperature dependence of the uptake?
(6) Do different fatty acids have the same kinetic parameters of
accumulation and assimilation? Is there competition between
fatty acids for the uptake mechanism?
MATERIALS AITO METHODS
Materials
Animals
Oi'sters of the genus and species Cvassostrea virginica were
collected from an estuary en the west coast of Florida north of Cedar
Key known as Shell Mound. An area of collection was chosen v/hich was
accessible without a boat and at mean low tide was covered witli two-
three inches of water. The experimental plot was sheltered froir. heavy
boat traffic and was exposed to a minimum of pollution due to the unpopu-
lated flrr'P around it. Animals cf 7 - 10 cm shell length (2.5 - 3.5 grams
soft tissue weight) were selected at low tide and onJy during stretches
of good weather so that there would be no effects due to large fresh
water influx and salinity change. The normal salinity for the area
ranged from 22 - 29 parts per thousand salt depending upon the tide. The
animals were brought back to the laboratory in plastic buckets covered
with wet canvas and were placed in a 20 gallon glass holding aquarium
eq'.npped with two Dynaflow circulating filters and an undergravel filtra-
tion apparatus. They were not fed in the holding tank and were used
within 72 hours after collection. Animals w^ere used from July through
M^y because those collected during the early summer were small and
gravid, frequently releasing eggs into the holding tanks or during the
uptake experir..;nts.
17
18
Chemicals
l^^l__-._.... -_.. ro lA^. _._ 1^
[l-"^ C]palraitic acid, [3- Cjstearic acid, [16-" Cjpalinitic acid,
and [7,8-"H]oleic acid were purchased from Schwarz-Maun. All non-
radioactive fatty acids vjcre reagent grade and were recrystalizcd before
use. Standard samples of phospholipid and neutral lipids for thin layer
chroniaLOgraphy (TLC) and fatty acid methyl ester standards for gas
liquid chromatography (GLC) were purchased froa Applied Science, Supelco
or Sigma Chemical.
Petroleum ether for extraction, column chromatography and TLC v.-gp
purchased from Eastman Chemical or City Chemical of New York, and nlass
distilled two times over potassium permanganate. It was separated into
30 - 60 C and 60 - 78 C boiling fractions and was stored in dark bottles.
Aquascl was purchased from New England Nuclear and spectr^il grade
toluene PPO-POPOJ? was made with reagents purchased from Sigipa Chemical ,
Chloroform and methanol for extractions v/ere purchased from Eastman
Chemical as analytical reagent grade solvents and were not redistilled
prior to use. Anhydrous diethyl ether was purchased from MallJnckrodt
and was not redistilled prior to use.
All other organic and incigsnic chemicals were analytical or
reagent grade.
S^ilanization of glassware
All glassware for uptake experiments, extraction, transporting and
storage of lipid material in aqueous or organic solvents was treated
with an aqueous silaniiiing rea-f-i-t, "Siliclad," purchased from Clay
Adams, Inc.
19
Column packings for GLC
EGSS-X and Apiezon-L column packings for the gas chromatography of
fatty acid methyl esters were purchased from Applied Science Labs.
Thin layer plates
Thin layer plates of Silica Gel 60 of 250 ym thickness on 20 x 20 cm
glass were obtained from E. Merck. Silica Gel G with no binder was
obtained from Applied Science and was spread on glass plates.
Column chroma tography
Specially prepared 400 mesh silicic acid for lipid column chromato-
graphy was purchased from Bio-Rad. Hi-Flosil, a silicic acid derivative
for rapid separation of lipid classes, was purchased from Applied Science.
Sea water collection and filtration
Sea water used in the uptake experiments was collected from the
Cedar Key estuary along witVi the oysters and transported to the laboratory
in 12-liter glass carboys or 5-gallon vinyl plastic containers. Before
use, the water was first filtered through a U^hatman ill paper under vacuum
to remove large particles and then filtered through a V-Tiatman GF/A glass
fiber filter of 0.45 ym porosity. The filtered sea water was stored in
g^ass at 4 c until used as an uptake medium. Sea water to be extracted
for background free fatty acid levels was saoipled as soon as the filtration
steps were completed.
Methods
Uptake ExperJHents
Closed shell experiments
The oysters to be used V7ere re:novcd from the holding tank and cleaned
of all epiphytic and epdzoic material with an oyster knife and a heavy
bristle brush. They were then rinsed clean of al] sand and left until
the shells were dry.
The labeled fatty acid was added to a small glass petri dish and the
earlier solvent, usually benzene, was removed with a stream of N„ gas.
A Teflon stirring bar was placed in the petri dish aiid the dish was
placed in a six-liter glass vessel. Four liters of bacteriologically
filtered sea v/ater containing 200 mg/liter of streptomycin sulfate was
added with stirring. The sea water was sampled by removing 1 ml alinuots
and counted in 10 ml of Aquasol. After the extracts reached a constant
specific activity, the animals were placed in the sea water, then
removed at various times and extracted.
In early experiments, extraction was carried out by a modified Bloor
method using a perchloric acid precipitation step followed by an ethanol-
ethtr (3:1) extraction (Bloor, 1928). This procedure is outlined in
Figure 3. In later experiments, a modified Bligh and Dyer
0-959) extraction was used. This ii'.volved homogenization of the whole
animal tissue in chloroform-methanol (2:2) followed by isolation of the
chloroform fraction (Figure 4). In either extraction method, an aliquot
of 1 li'l of the cthanol-ether or 200 yl of the chloroform extract was
added to Aquasol and count ci.
21
Count
1 ml aliquot
20 ml II 0
Add 70%
HCIO, to make 0.6 M
Oyster (remove from shell)
V
Wash in 100 ml of sea water saturated with
palmitate
I
Weigh (to nearest 0.1 gram)
~>
Homogenize 15 seconds in VJaring blendor
i
Decant into 25 x 150 mm centrifuge tubes with
2 rinses (volume '^ 40 ml)
Allow protein to precipitate (5 minutes at room
temperature)
Spin in lEC centrifuge 2000 rpm :l IC lainute;
Muscle mat (discard)
Supernatant (discaid)
Pellet
Add 40 ml Bloor Reagent: FtOK-ether, 3:1
T
Allow protein to percipitrte, centrifuge (lEC)
2000 rpm x 10 minutes
Count
1 m.l aliquot
supernatant'
pellet (discard)
Figure 3. Extraction uith Adapted Bloor Method.
22
Count <i.
1 ml aliquot
10 ml CHCl,
20 ml MeOH
10 ml CHCl
Oysters (3-4, remove from shell)
I
Wash in 100 ml of sea water saturated with
fattv acid
Weigh oysters on pan balance to nearest 0.1 g
(should be near 10.0 grams)
i
Adjust weight with HO to equal 10.0 grams
Homogenize 90 seconds in Waring blendor at
slow speed
HoT.cgenize 30 seconds in Waring blendor at
high speed
Retentatc
(discard)
Vacu'om filter through Whatman #1 paper
V
Filtrate (allow to settle into two layers,
record volumes)
Count
200 yl aliquot
Aqueous methanol
CHCl,
Count 200 111 aliquot. Retain for incorporation
studies.
Figure 4. Extiaccion with Adapted Bligh and Dyer Method
23
Open shel3 experiment s
For those uptake expcrinents in which concentration dependence and
competition were investigated, a modified procedure was used in order to
eliminate variations in the data caused by any periodicity of vcJ.ve
opening and closing by the experimental animals. The upper valve was
removed by wedging the hinge and then carefully separating the adductor
muscle from its upper shell insertion. Only those animals in which no
traumatic tissue damage was evident were used in these experiments.
Continuation of regular heart beat and a non-ruptured pericardial cavity
were used as a test of viability and successful removal of the Gpu-r shell.
The animals were rinsed in sea water and then placed in the vessel con-
taining 4 liters of filtered sea water. At zero time r.-ie. labeled fatty
acid and any competing fatty acid dissolved in ethanol were added to
4 liters of sea water below the surface of the vortex created by a .stirring
bar, ensuring that the ethanol and the fcxety a.ici were ux',uer3cd rapidly
throughout the medium. In these experiments, the carrier ethanol concen-
tration never exceeded 5 parts per thousand and no effects of the solvent
were ever seen. The sea water was sampled by removing 1 ml aliquots at
various times and counting in Aquasol. Samples were also taken and
filtered through 0.45 pm filter and counted in Aquasol to determine
whether the labeled fatty acid aggregated or was adsorbed on aggregated
material.
Celite uptake experiment
The uptake of fatty acids adsorled on celite was studied using
Johns-MansvJlle celite sieved to approximately 50 ym size particles. The
fatty acid in appropriate concentration in ether solution was added to
24
the dried celite and the solvent removed under vacuum with a Rinco
evaporator. This process of solvent addition and removal was repeated
3 times and the celite dried under N to remove traces of remaining
solvents. The celite bound fatty acid was then added with continuous
stirring to the sea water containing the experimental animals and after
suitable time periods, samples Xi?ere taken and tlie procedure outlined in
Figure 3 or k followed. The sea water was sampled both unf liter ed and
after O.-'iS ym filtration to determine the concentration of free fatty
acids, and therefore, the degree of dissociation of the f,-;tty acid from
the celite particles.
Temperature dependent uptake experiments
The temperature of the sea water solution was maintained using a
copper-coil cooled/heated water reservoir around the 6 Jittr gla^s ve£?^=l.
The cooling or heating water in the coil was circulated from a Forma
Scientific vzater bath. The temperature of the sea water was thermo-
statically maintained with + 1 C of th^ desired temperature. The studies
of uptake were the same as described previously.
Tarnover experiment
The animals were prepared as for the uptake experiments b^L the
shells were not removed. The oysters were placed in a glass vessel with
4 liters of filtered sea water. Seven mg of sodium [\]acetatc (5 mCi)
was acMed to the sea water. After IS hours, the animals were removed,
washed in sea water, and placed in a glass vessel with 4 liters of
non-radionctive sea v.'ater. Groups of 3 oysters were removed at 0.5, 1.0,
2.0, 3.0, and 5.0 hours and extracted by the chloroform-methanol method.
25
Aliquots cf the extract were separated on TLC plates and counted and
quantitated.
Resolution of Lipids
Thin layer chromatography
The neutral lipid classes were resolved by thin layer chrorr.atography
techniques and identified by comparison with standard compounds. The
250 ym si2ica gel plates were divided into 2 cm channels and activated by
heating for 30 minutes at 120°C. Lipid extracts (100 or 200 pi) were
applied with an Oxford pipetter 1,5 cm from the bottom of the plate and
the solvent evaporated with a stream of hot air. The plates were developed
in a FE/EE/HOAc (petroleum ether (30 - 60°C) /die thyl ether/acetic acid)
.'Jolvc-mt (90/10/1) for approximately 1 hour. The solvent was removed
with a ftream of air and the material on the plate ur.j. visualized with
either iodine vapor or by charring v/ith sulfuric acid (Mangold,
1960). This TLC solvent system completely resolved the neutral lipid
classes of Cvassostvea and the sea water extracts into sterols, tri-
glycerides, alko'l diglycerides, wax esters and sterol esters.
The phospholipid classes were resolved o-; silica gel plates
activated for 30 minutes az 80°C, and developed in a chloroforra/methanol/
water solvent (55/25/4) for approximately 80 - 100 minutes (Wagner,
19G1). The plates were channeled and the extracts spotted in the same
manner as the neutraj lipi'.ds.
A letter separation of the phospholipids could be achieved v/hen the
neutral lipids were first removed from the extract by column chronato-
graphy over a 1 x 10 cm Hi~Flosll column. The extract, in chloroform,
was applied at the top and all the neutral lipids eluted '.jith 2 column
26
volumes of CHC1„. The phospholipids vjere then stripped from the column
bed with methanol. After removing the methanol in flash evaporator,
the extract was taken up into chloroform, spotted on a TLC plate, and
run m the polar lipid TLC system described previously.
For complete resolution of phospholipids, a two-dimensional method
v/as used in which the plate was developed in chloroform/methanol/water/
28% aqueous ammonia (130/70/8/0.5) in one direction and chloroform/
acetone/rcethanol/acetic acid/water (50/20/10/10/5) in the 90*^ direction
(Parsons and Patton, 1967).
Table 3 lists the visualization reagents which wore employed in
the identification of the neutral anc plicspho.ipid compounds.
These reagents, together with a saponifiLcation step for esterified
compounds (Stahl, 1969), permitted the idantif ication of the lipids found
i~i the oysters.
Quant:! ration of lipid material
The lipids following separation by thin layer chromatography were
quantitaced using the method of Amenta (1964). The lipid on the TLC
plate was visualized with I^ vapor and scraped into glass tubes; ] cr 2
ml of a 8.5 yM solution of potassium dichromate in concentrated sulfuric
acid were added. Tht^ tube was stoppered and heated at 80 - 100°C for
45 minutes in a water bath with constant agitation. The tubes were
removed, allowed to cool, and centrifuged in a clinical centrifuge to
pellet Che silicic acid. A 0.5 ml aliquot of the supernatant was
removed, diluted with 10 ml of distilled water, and stirred to mix
thoroughly. The absorbance of this solution was determined ac 350 nm,
comparing against a water blank. The difference in absorbance between a
Tablo 3. Visualization Reagents for TLC.
Reagents
Function
Reference
I^ Vapor
Chromic Acid-
Sulfur ic Acid
General Screen
General Screen
Bettschart and
Fluck, 1956
Bertetti,
195A
Rhodamine B
Ninhydrin-Butanol
Chromic Acid-
Glacial Acetic Acid
(1:1)
Ammonium Molybdate
General Screen
Amino-Phospholipid
and Glycolipids
Containing Glucosamine
Cholesterol
Cholesterol Esters
Phospholipids
Kaufmann and
Budwig, 1951
Fahmy
et al., 3961
Mi.chalec,
1956
Hanes and
Isherwood,
1949
Kydioxyl /uniue-
Fcrric Chloride
Esterifieu C.irboxyiic
Acids
Whit taker and
Wijesuiidera,
1952
28
standard tube and a sample tube was compared to curves for cholesterol,
tripalmitin, cholesteryl stcarate, dimyristyl phosphatidyl choline, and
palmitic acid.
Scintillation countiuR
The aqueous samples of sea water, wash, and filtered sea water from
the uptake experiments were counted in Aquasol (1 ul aqueous sample added
to 10 ml scintillant) . The chloroform and methanol layers of the extracted
material were counted in Aquasol at 200 lij /lO ;uL to reduce quenchia^ of
the organic solvents. All samples were counted in a sub-ambient Packard
o
TricarD at 0 C and compared tu suitable standards. The doubit: label
experiments were counted in a refrigerated Nuclear Chicago ccimter in the
double label mode.
The radioactive lipids, once separated on TLC plates, were either
counted directly in a Packard TLC radlosc;.'-iner or the lipids were scraped
off the plates directly into 5 ml of Toluene FOPC'P ana counted in a
refiigerated Packard Tricarb. The efficiency of this method is much less
than reported by others (Kritchevsky and Malhotra, 1970) but it is much
simpler than a solvent extraction-Aquasol counting procedure.
All scintillation counting work was corrected for back^p-oupd and
counting efficiency by coincidence counting with [ Cjf.oJueae,
14 3
[ C]benzoate, and [ H]water standards purchased from Packard Instruments
and diluted as required.
Fatty Acid Methylation — GC Separation
P reparation of met hyl er t ers
The fatty acids were methylated according to the method of Stoffei
29
et al. (1959). To tlie fatty acid samples separated by thin layer
chromatography were added 4 ml of 0.24 N HCl In methanol and 0.5 ml of
dry benzene. The solution was refluxed at 80 - 100*^C for 2 hours in a
ground glass apparatus fitted with a CaCl drying tube. The reaction
mixture was cooled to room temperature and 9 ml of H 0 were added to
quench the reaction. The aqueous solution was extracted 3 times with
petroleum ether (30 - 60°C) and this extract was dried over Na SO and
2 4
NaHCO^. Tlie petroleum ether was added to a sublimation apparatus (a side
arm test tube fitted with a cold finger) and evaporated. Then tne fatty
acid methyl esters were sublimed in 200 ym vacuum and at 60 + 2°C. The
methyl esters vrere rinsed with hexane from the cold finger into a small
vial and were injected into the GC.
A second procedure (Hoshi et al. , 1973) was employed for methylation
at room temperature. It required 0.2 ml of the sample fatty acid in
chloroform, 0.2 ml of 20 mM cupric acetate in methanol and 1.0 ml of
0.5 N HCl in methanol. The solution was allowed to react at room
temperature for 30 minutes and then, after the addition of 0.4 ml II 0,
was extracted 3 times with 2 ml of petroleum ether (30 - 60°C) . The
extracts were pooled, washed with HO, evaporated to dryness, and
redissolved in hexane before injection into the GC.
Saponif ica tion and methylaticn
The direct saponification and methylation of fatty acids in the
lipid extracts were performed using a modification of uhe procedure of
Christopherson and Glass (1971). The lipid extracts were added to Teflon-
capped tubes and the solvent evsporated to dryness with N, gas. Five mi
of 2 M potassium hydroxide in methanol solution were added and heated at
30
AO - 50 C for 30 minutes. After the addition of 6 ml of water, the
solution was extracted 2 titres with 5 ml of petroleum ether (30 - 60°C) .
The ether solutions were pooled, evaporated to dryness, taken up in
200 - 500 "^1 of hexane, and stored in small 1 ml vials with Teflon-lined
screw caps. Aliquots (5 - 10 yl) of this hexane solution were injected
into the gas chromatograph.
Gas Chromatography
The fatty acid methyl esters were run on 2 different column systems
in a Beckman GC-65 gas chromatograph with N^ as the carrier gas and dual
hydrogen flame detector. An organo-silicone polymer, EGSS-X, at a 10
percent loading on 100/120 Gas Chrom P-Support in 2 m x 4 mm glass column
was run isochermally at 1S0*^C. This column resolved the 16-C and 18-C
series of fatty acid esters, hut even at its maximum temperature the
higher boiling poly-unsaturated acid esters were not eluted. Therefore,
initial experiments were run on dual Apiezon-L columns at a 2,25 percent
loading on 100/120 Gas Chrom G in 1.3 m x 4 mm glass columns. The gas
chromatograph was programmed from 170 - 275°C at 1. 5°C/miinite at which
temperature the higher boiling esters were eluted.
The later determinations were done on an EGSS-X column run isothermally
at 174 C with a 45 ml/minute flow rate and at 190°C with a 60 ml/minute
flow rate. At 174 C EGSS-X columns resolve lower boiling fatty acids and
the 18 series; at 190 C the long chain unsaturated acids are eluted. This
column does not suffer from large bleed rates that Apiezon columns show
at higher temperatures, therefore, almost a].l acids reported in 10-C -
22-C range can be resolved without difficulty (Applied Science, 1973).
DATA AND DISCUSSION
' Lipids and Free Fatty Acids in Sea Water
The sea water of the Shell Mound estuary was sampled in 8 liter
quantities for determination of total lipids, compound lipids, and
specific free fatty acids during the spring, summer, and fall. The water
was extracted as described in the section on methods and fractionated by
thin layer chromatography. The results of the neutral and phospholipid
chromatography of the June 21, 1974, October 31, 1974, and the March 31,
1975 samples appear in Figures 5 and 6. The absence of phospholipids
from the June ?1 and March 31 extracts and their presence in the chloro-
form extract of the October 31 sample can be attributed to the i:se of
petroleum ether (30 - 60°C) for their extraction. Jeffrey has showi that
a complete polar lipid extraction can be achieved only with chloroform
(Jeffrey, 1970). However, because we were interested primarily in the
uptake of free fatty acids, the use of petroleum ether x,;as justifipd.
Preliminary experiments with *C ]abeled fatty acid revealed that better
than 90 percent extraction of the label could be effected with 1 extraction
step v.'lth petroleum ether (30 -- 6o"c) and 3 subsequent washes of the
extract with 2 N HCI .
By comparison of. the lipid extracts wj tri knovm standards, those lipid
classes whicl) are separated by TLC can be id-titified and quantitated by
the raethods previously described. The results appear in Table 4. For the
June 21 extract the majority of the lipid appeals to be in the free fatt^
:v
31
- Cholesterol
5 -Phosphatidyl Ethanolamine
- Phosphatidyl Choline
Lyso-phosphatidyl
Choline
Figure 5. Separation of Polar Lipids in Sea Water Extract
s.
Sea \;ater was extracted with petroleum ether (June 21) or
chloroform (October 31) and 200 yl aliquots run on the polar
lipid TLC system. 1, BFSW (bacterially filtered sea xcater)
from Oct. 31; 2, NBFSW (non-bacterially filtered sea v.oter)
from Jur.e 21; 3, BFSW June 21; 4, cholesterol standard;
5, phospholipid standard with stanrlards listed on the right
margin. Dotted line at the top: solvent front.
^:)
\
a
r
u
o
o
Q
o
o
O
^-v-^
/
O
O
8
O
O
o
r.\
O
0
ZD
o
i / \
- Sterol Ester
- Fatty Acid Ester
- Triglycei^ide
- Free Fatty Acid
- Diglyceride
- Sterol
- Polar Lipids
Figure 6> Separation of Neutral Lipids in Sea Water Extracts.
Sea water v,as extracted with petroleum ether and 2C0 yl
aliquots run on the neutral lipid TLC system. 1, NBFSW
June 21; 1, BFSW from June ?.l ; '^ , BFSW from March 31;
4, standard neutral lipid mixture with compon'jnts listed
in the right margin. Solid line at the top was the solvent
front.
Table 4. Concentrations of Extractable Specific Lipids in the Sea Water
Collected on June 21, 1974 (Extract A) and Msrch 31, 1975
(Extract B) .
Rf'
0.04
0.06
0.12
0.21
0.36
0.65
0.88
0.94
Lipid Class
Monoglyceride
Sterol
Diglyceride
Free Fatty Acid
Triglyceride
Alkyl Diglyceride
Sterol Testers
Hydrocarbons
Total
Concentr;
ations
in yg
in
/I
Sea Water
Extract
A
Extract R
Trace
Trace
14
32
Trace
Trace
77
36
31
4?
3(.
13
104
62
53
28i
'Relative migration of lipid class on a neutral lipid chromato-
graphic system relative to the solvent front migration.
35
acid and hydrocarbon fractions; together they comprise greater than
50 percent of the total lipid. The concentration of the free fatty acid,
77 Jjg/liter, compares favorably with previous determinations reported in
the introduction. For the June 21 extract, the free fatty acids were
eluted from the silica gel and methylated. The methyl esters were run
on the gas chromatograph with the results shown in Figure 7. The fatty
acid distribution is similar to that obtained by Testerman (1972).
The percentage of each fatty acid present, corrected for differences
in detector sensitivity, appears in Table 5. From these data, the pre-
dominant fatty acid in the sea water at Shell Mound appears to be
palmitic acid. The notable absence in our work of those long chain
unsaturated acids, 18:3, 18:4, 20:1, 20:2 found by others (Jeffrey, 1970),
can be attributed to the complete removal of all algae and bacteria prior
to extraction, for these acids are characteristic of such organisms.
In the sea water extracts from Shell Mound, the fatty acids which
are characterized are free by definition of the experimental methods used.
The saponification step, used by others, has been intentionally eliminated
from the extraction-separation-methylation steps so that only those fatty
acids which are free in solution are extracted. The inclusion of a
saponification step before niethylation by Testerman, Jeffrey, and others
was intended to break up any lipid organic aggregates in the sea water so
that complete extraction might be effected.
The data in Table 4 indicate that large amounts of free fatty
acids are present in the sea vrater at Shell Mound and that these might be
expected to be readily available for removal by any animal possessing an
uptake system which functions at these naturally occurring concentrations.
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38
Table 5. The Free Fatty Acids in the June 21 Sea Water Extraction.
The retention time and percent composition of the fatty acid methyl
esters are from GC run Figure 7 and corrected for detector response.
Carbon Number ^n^Minutes"''^ Percent Composition
C-12 2.2
C-14 3.5
C-16 6.1
C-18 10.8
C-18:l 12.1
C-18: 2 16.0
C-20:0 20.2
27.6
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2.8
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39
Uptake of Palmitic Acid
The ability of oysters to remove palmitic acid from natural sea
water solutions was investigated with [l-""" *C]palnitate at a concentration
of 2.8 X 10 M. The background concentrations of total lipids and free
fatty acids were determined and the specific activity of the palmitate
was computed from the a-nount of isotope and carrier used. In each set
of experiments sea water from the same sample was used throughout to
minimize any differences in salinity which might have affected the
uptake. Stephens has ^hov.m that the salinity of the sea water drastically
affects the uptake of amino acids by coelenterates (Stephens, 1963).
Natural sea water was chosen so that any trace elements or dissolved
organics which are not present in artificial mixtures, but which may
affect uptake processes, would be present. V/ith artificial salts, in
the quantities needed to make a 28 parts pej: thousand salt sulucion,
organic contaminant-.s will be present in large concentrations compared to
10 M fatty acids. Even reagent grade salts could contain significant
quantities of non-extractable lipid and hydrocarbon impurities.
In the experiments on lipid uptake by oysters, the lipid label might
be expected to adhere to the mucus and the soft tissues of the animals.
A satislactory method of removing this adventitiously adsorbed material
had to be developed. In the early work with hydrocarbon uptake by Lee
et al. (1972), a methanol v;ash was employed, but we found this severely
dehydrated the animals and could possibly cause the removal of more than
just adsorbed material. A wash procedure in sea v.'ater saturated V7ith
the experimental fatty acia was found to exchange effectively any
simply adsorbed material (see Figi;re 8). The loss of label could then
be monitored by sampling the- wash solution at 30 to 60 minutes. In all
40
AO-^
30
e
p.
20 -
I " /
20
40
"V—
60
80
100
Time in Minutes
Figure S. Diffusion of Adsorbed Labeled Fatty Acid into a Sea Water
Wash Saturated with Unlabeled Palmitate.
Animals labeled with palmitate for 240 minutes were placed
in 100 ml of filtered sea water containing a saturating
amount of palmitate. The sea v/ater was sampled in 1 ml
aliquots and counted in 10 ml of Aquasol. Aiiimals were
labeled with 10 pCi •'-'^C palmitate at a concentration of
2.8 X 1U-' M.
Al
uptake experiments such a wash step was employed and found to be
satisfactory.
In order to monitor fatty acid uptake by the oyster, procedures
involving lipid extraction were used. Experiments with tissue solubili-
zers proved unsatisfactory with animals as large as oysters, since their
weight (3 grams average in experimental animals) is above the upper
limits of the tissue sample weight for such alkaline solubilizers.
Although the work with nereid and pogonophoran species utilized such a
digestion step to sample single animals or groups of animals, the oysters
had to be extracted. Preliminary experiments with petroleum ether (30 -
60 C) extraction techniques on aqueous homogenates proved unsuccessful
due to the stable emulsion formed at the organic-water interface. After
using a step involving perchloric acid, the precipitated protein could
be pelleted along with included lipid materiel. This pellet could then
be isolated and extracted with ethanol-ether (3:1). The lipids were
solubilized and the protein remained as a precipitate. Using tracer
techniques of labeled fatty acids, this method of Bloor (1928) was shown
to be 75 percent effective in extracting lipids from the oyster aqueous
homogenate. The results of an uptake experiment at a palmitate concen-
tration of 2.8 X 10 M using the saturated wash step and the Bloor
extraction method appear in Figure 9. The major loss of label from the
sea water occurs in the first 60 minutes and is coincident with the
appearance of the label in the lipid extract. The loss of labeled
material from sea water was phown to be a function of the living animals
and was not due to adsorption onto the shells or the walls of the glass
vessel by carrying out a blank experiment with a similar weight of oyster
shells cleaned and washed according to the methods for whole animals
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(see Figure 10). In this control experiment less than 10 percent of the
label V7as removed from the water.
The effect of 200 mM sodium cyanide on the uptake of 2. 8 x 10~^ M
palmitate was investigated. As seen in the data in Figure 11 , the
radioactivity in the lipid extract remained very low and the label in
sea water remained constant, indicating that the background adr.orption
of lipid onto the animals in the absence of uptake was indeed small.
The animals were not killed by the cyanide for at least 2 hours but
their respiration was severely inhibited. A large concentration of
cyanide was used because of the oyster's kno^^i ability to carry on
anaerobic metabolism (Hammen, 1969).
The Bloor method of extraction did not permit the quantitation of
the lipid classes because of the hydrolysis and esterification that
occurred in the acidic ethanol/ethsr extraction step. A chloroform--
methanol extraction (Bligh and Dyer, 1959) as described in the methods
section v.'as therefore utilized for all further uptake investigations.
The variability of the amount of radioactivity in the sea water at
time zero in Figures 9 and 10 was ascribable to an artifact in the addi-
tion of Che labeled acid to the sea water. At first the labeled fatty
acid, dissolved in ether or benzene, was added to a glass petri dish.
The solvent was removed with nitrogen, and the petri dish was placed into
the reaction vessel. The amount of label that dissolved in the sea water
was dependent upon the temperature, the solubility of the fatty acid,
and the degree of agitation of the solution. Of these variables, the
agitation was least reliable, so a method involving direct addition of
the labeled fatty acid dissolved in ethanol was devised. This was shown
in preliminary experiments to be a simple and most reliable method of
45
e
B
a
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X
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2.0
0.5
1.0
30 60
Time in Minutes
— r-
90
120
Figure 10. Removal of C Fatty Acid by Background Absorption onto
Shells and Class Surfaces.
The loss of labeled fatty acid from the sea water in a
vessel containing 2,8 x 10~^ M palmitate with 10 uCi ^-'^C
isocope and the shells of the same number of aninajs as
■normally used in the uptake experiments wa? plotted against
time. The shells were washed according to the methods
used for whole live animals. *
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dispersins the acid. The problems of variability of initial label con-
centration in sea water were reduced significantly without any side
effects of the ethanol on the animals.
From the early series of experiments involving long-term uptake of
up to 3 to 6 hours, it was apparent that the uptake maximum occurred at
about 1 hour with a subsequent leveling off of the radioactivity in the
sea water and lipid extract pools. That this leveling off was due to
the removal of most of the free fatty acids by the animals was shown in
a repeated pulse experiment in which the labeled fatty acid, dissolved
in ethanol, at a concentration of 2.8 x 10 M (palmitate) was added at
time zero and at 180 minutes. The results, shown in Figure 12, indicate
that the labeled fatty acid concentration in the sea water decreases
rapidly in the first 3 hours, coincident with the appearance of label in
the lipid extracts of the animals. After the second pulse at 180 minutes,
the fatty acid level in the sea water again decreases with a concomitant
increase of labeled acid in the lipid extracts. The regular differences
in the coiints in the lipid extracts were caused by the periodicity of
valve opening and closing in the animal's normal feeding cycle, but the
data shox-7 that during the first 2 hours almost all the label is removed.
The presence of CO in the sea water, shov/n in Figure 12, Indicates
that the animals were metabolizing some, at least, of the fatty acid
removed. The gradual increase in slope after the second addition of
labeled fatty acid may indicate that the breakdo;>m of free fatty acid
is proportional to the amount of the fatty acid removed.
To avoid irregular valve opening, a method of synchronization was
employed. The best method took advantage of the normal behavior of
animalj exposed to air during the tidal cycle. When experimental
49
(X
o
60
•H
60
e
•--^
6
a
Time in Minutes
Figure 12. The Uptake of Palmitate, Double Addition of Label.
The radioactivity in aliquot.<^ of the sea v/ater (B) , the
lipid extract (A), and CO2 in an aliquot of sea water (C)
v.rere plotted aj^ainst time. The concentration of palmitate
. in the sea water was 2.8 x lO"? M after the first addition
at time zero and 2.8 x 10-7 >; after the second addition. A
total of 20 uCi of l^c isotope was used; 10 yCi at each
addition. The label was added in ethanol. l'^*C02 was
counted after trapping in hyamine hydroxide and adding to
Aquasol. The lipid was extracted by the chloroform/methanol
method and ploLted as the dpm/rjg oyster tissue in each
sample.
50
animals were removed from the holding tank, cleaned as usual, and left
to dry in the air for 3 hours, then placed in the radioactive free fatty
acid containing sea water, the shells opened almost immediately. Opening
in the first few minutes is essential to the determination of initial
rates of uptake necessary for kinetic determinations. The variability
of the data even after such a synchronisation attempt by an out-of -water
phase necessitated experiments in which the top shells were removed.
When the upper shell was removed carefully and the muscle, gill,
mantle, and pericardial tissue were not traumatized, the uptake of label
into the aniimal was more reproducible (see Figure 13). The maximum
labeling of the lipid pools was linear with time and occurred during the
first 90 - 120 minutes.
The temperature dependence of the uptake process was investigated
using the experimental apparatus described in the methods section. The
temperature dependence of palmitate uptake at a 2.8 x lO""'' M concentra-
tion was investigated at temperatures of 20, 25, 30, and 35°C. The
results appear in Figure 14 as the average uptake for two experiments at
each temperature. The inverse dependence of the uptake on temperature
which is seen in the experiments is similar to what has been reported
before in uptake experiments on other marine animals (Shick, 1975). The
uptake of fatty acid at 20°C was virtually zero with the response of the
animals being a decreased shell opening cycle. The temperature dependent
uptake therefore may represent a physiological response of the animals
to temperature and not a response of the uptake machinery to temperature.
Celite Uptake Experiments
The major assimilauory pathway utilized by the oyster is filter
feeding via the ciliary apparatus o^ the gills. UTiil e uptake of free
51
50
2-
-40
t-H
(5
G
><
ro
I
O
r-1
- 30
20
40
60
80
20
- 10
100
s
■u
to
Time in Minutes
Figure 13. The Uptake of Palmitic Acid by Open Shell Animals.
The loss of labeled fatty acid from the sea water (E) and
the appearance of label in the lipid extract (A) was plotted
against time of exposure. Animals with the upper valve
removed were placed in 4 liters of sea water with a paJmitate
concentration of 2.8 x IQ-^ M and containing 10 pCi total
C isotope. The lipids were extracted by the chloroform/
methanol method and the dpm/rag wet weight of the oysters
plotted.
52
•H
OJ
^3
3 5°C
Time in Minutes
Figure 14. Temperature Dependent Uptake of Pairaitate.
The radioactivity in the lipid extract of the animals was
p]otted for three different temperatures. The concentration
of palmitate was 2.8 x 10"'' M with 10 yCi total -'-''"'C isotope
in all experiments. Each point was the average of two experi-
ments at each temperature. The results for 20°C were
negative to 300 minutes. The lipids were extracted with
chiorof orm/methanol.
fatty acids can be established, it may represent merely the removal of
fatty acid particles through prior adsorption on a mucus thread followed
by the ciliary transport of this thread through the digestive apparaf.'s.
In the autoradiographic work by Pequignat (1972) , the labeled amino
acids which were taken up from the sea water by Mytilus edulis were first
found in the gill, the mantle, and the foot. Only after a much longer
period of time were silver grains on the photographic emulsions found in
positions corresponding to the digestive tract and to the mucus secretions
on the gills. In order to establish the time sequence of particulate
filtration in oysters, a preliminary experiment with celite of 50 ym
particulate size was used. An aniline dye, oil red 0, in ether solution
was adsorbed onto the celite particles by successive washes with the
ether solution followed by evaporation of the solvent. Fifty mg of
dyed particles were added to A liters of sea water and the evtent of untr-tke
determined by visual inspection before and after dissection of the animals.
The presence of red particles was noted on the external surfaces and in
the digestive tract. Aliquots (5 ml) of the sea water in v/hich the
particles were suspended were extracted with petroleum ether and the
absorbance at 525 nm (the maximum for oil red 0) determined. The results,
shovm in Table 6, indicate that particles are adsorbed onto the mucus
' thread within the first 30 minutes and into the digestive tract after 90
minutes. Because oil red 0 is not digested by the animal, it is sorted
and appears in the feces after 90 - 120 minutes.
Knowing that celite particles are removed from sea water by oysters,
the uptake of celite-adsorbed [ Clpalr.iitate was investigated. Ten yCi
. 14
or C labeled fatty acid was adsorbed onto 50 mg of 50 yir celite
particles with successive etliyl ethtr evaporations as described in the
54
Table 6. Localization of Oil Red 0 Celite Particles Removed t'rcm Sea
Water by Experimental Animals.
T T- ^. c Time of First Absorbance (525 nm )
Localization or . ^
^ ,.. 13 .. , Appearance of Pet Ether Extract
Celite Particles ,,,. .
(Minutes) oi Sea Water
Sea Water 0 0. 2S9
Mucus Thread 30 0.232
Oral Cavity 60 0.291
Digestive Tract 90 0.218
Anus, Feces 90-120 0.147
Five ml sea water extracted with petroleum ether 30 - 60°C and
read into a visible spectrophotoiaeter.
55
methods section. The uptake of this labeled celite was investigated
with whole animals. Figure 15 shews that the total radioactivity in the
sea water decreases as the incorporation into the lipid extract increases,
with the exception that the appearance of the label is delayed by some
A5 minutes when compared with the uptake of similar concentrations of
freely soluble palmitate at 2.8 x 10~ M. This delay has been seen in
every celite particle uptake experiment run with oystera. It represents
a delay in the incorporation of labeled acid particles into the animal
by the filter feeding apparatus when compared to the uptake of non-
particulate fatty acid. These results are, therefore, similar to
Pequignat's findings on the uptake of amino acids by hhjtilus edulis, the
label appearing in the gut much later than that which appears in the soft
tissues.
The concentration of free acids in the sta water was determiaed by
the dpra/ml in a 0.45 pm GF/A filtered aliquot. From Figure 15 there
appears to be a constant amount of radioactivity in the filtrate indi-
cating only minor dissociation of the particle-bound fatty acid into
free acid.
The uptake of celite-adsorbed palmitate at 2.8 x 10~^ M was also
investigated using the open shell animals (Figure 16). There is a dif-
ference between their accumulation of Isbel and that in the whole
animal experiment. The organism can remove the lab^l very efficiently
and at a linear rate up to 90 minutes. If this celite uptake is compared
to the uptake of 2.8 x 10 M palmitate for open shell animals (Figure 17),
the rates (slopes) of uptake are different. The use of a concentration
factor (Taylor, 1969) allows comparison of the tv;o different sea water
concentrations as dpm/ral of sea water/Vulpji/mg of animal tissue in the
56
2-
6
S 1 4
.30
ft
\
7'
•(c)
/
60
i
120
180
2A0
40
20
.10
u
CO
•H
E
Time in Minutes
{"igure 15. The Uptake of Celite-aclsorbed Palmitate.
The total radioactivity in a 1 ml aliquot of the sea water
(B) , a 1 ml aliquot of 0.45 ym filtered sea water (C) , and
200 yl of the chloroform extract of the animals (A) was
plotted against tir.e. The concentration of palmitate used
to prepare the 50 h= celite was 2.8 x 10"'' M. Three animals
were extracted at each point.
s?
10
50
£
I
o
8-
2.
^
40
•30
(A)
\
^:
4
/
/
■•(c)
20
40
— r-
60
80
to
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CO
e
~e
ex
X)
-20
^10
100
Time in Minutes
Figure 16. The Uptake of Celite-adsorbed Palrnitate, Open Shell Animals.
The radioactivity in 1 nl aliquots of the sea water (B) ,
1 ml 0.45 Uui filtered aliquots of the sea water (C) , and
aliquots of the chloroform extract (A) were plotted against
time. The ccnceixtrai-ion of palmitate used to prepare the
. celite was 2.8 x 10"' M. The animals were added after the
removal of the upper valve. Three animals ware sampled at
each point.
M
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60
chloroform extract. From Figure 17, the cellte uptake for open she]]
animals occurred at a faster rate than the uptake for whole animals. The
uptake by open shelled oysters is facilitated by the celite particles
dropping out of circulation in the glass beaker and onto the animals.
The fatty acids on the celite could then be exchanged from particle to
animal either in a mucus thread or across the water-tissue surface. This
process v7ould not and does not occur in whole animals where the movement
(by ciliary currents) of celite containing sea water through the shell
would bring the particles into contact with the filtering apparatus of
the gill.
The comparison of the uptake of free stearic acid and celite-bound
stearate by open shell animals is shown in Figure 18. The rates of
uptake are much lower than those for palmitate, but the celite-adsorbed
label is removed at a faster rate than free stc^.r-iite. The explanation
of these results would parallel that for palmitate; the rate of uptake
is enhanced due to particulate aggregates settling out of solution onto
the animals.
Concentration Dependent Uptake —
Kinetic-Parameters of Uptake
4
The concentration dependent uptake process V7as investigated with
14
open-shell animals and C labelled palmitic, stearic, and oleic acids.
The incorporation of [ Cjpalmitate and [ C]stearate into the lipid
extracts are plotted in dpm/min/mg wet weight as a function of the time
after uptake. The lines vjere computer plotted by least squares. See
Figures 19 and 20. The slopes of the plots of the initial rate of uptake
is plotted versur, concentration. Figures 21 and 22, the saturation p]ots
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Figure 21. The Concentration Dcp2ndent Rate of Uptake of Palmitate.
The initial rate of uptake determined from the slopes of
Figure 19 were plotted against the concentration of palmitate
in the experiments. The animals had the upper shell removed
prior to addition to che sea water.
68
0
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g
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4-1
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Figure 22. The Concentration Dependent Rate of Uptake of Stearate.
The initial rates of uptake determined from the slopes of
Figure 20 wereplotted against the concentration of stearate
3.n 5 experiments. The animals had the upper shell removed
prior to placement in the sea water..
69
for palmitate and stearate, show similar saturations at low concentra-
tions, then a sudden burst in the uptake rate appears at 3.0 or
&.0 X 10 M. This is probably due to self-aggregation of the fattj'
acids at the elevated concentrations promoting either an enhanced rate
due to large particle effects or due to generation of particles large
enough to permit the animals to filter them. The increased uptake rate
is seen in conjunction with increased turbidity of the sea water solu-
tion. The same concentration effect was seen by Testerman (1972) in
his experiments with fatty acid uptake. From his experimental work with
artificial sea water as a medium, he found the raicellar concentration of
palmitate to be about 5 x 10 M. In the experiments with natural sea
water reported here the micellar concentration is about 7.0 x 10 M.
The difference in the two figures emphasizes the importance of considering
thf contribution of other fatty acids in sea water when investigating
uptake rates.
The plots of the velocity-concentration data for palmitate and
stearate treated by the Lineweaver-Burk reciprocal method yield straight
lines. Figure 23, the palmitate plot for all data points below
6.0 X 10 M, i.e., below the aggregation concentrations, has a y
intercept. Km of 5.0 x 10 M, and a maximal velocity of 0.78 dpm/rag/mln.
If this rate is converted to the actual concentration of palmitate removed,
the rate becomes 2.3 pmoles/gram/hr . For stearate (Figure 24) the Km
is 0.59 X 10 and the maximal rate of uptake is 0.53 dpm/iag/mln. The
rate of uptake of stearate expressed in molar terms becomes 1.9 pmole/
gram/hr. These figures for the Km relate to the sea water concentrations
of the acids in natural coastal waters. From the data at Shell Mound,
the ambient concentrations of the acids in sea water are 1.1 x 10 M
IC'
8-
/
/
•y
4.
2 : : y
12
16
1
b
Figure 23. Lineweaver-Burk Transformation of Palmitate Uptake Data.
"The initial rates of uptake for 5 concentrations of palmi-
tate were plotted by the double reciprocal method. The
maximum velocity was determined from the y-intercept and
the Kra for the uptake process from the slope (V = dpm/mg
wet weipht/min) (S = 10"'' M omitting the point at
8 X 10-7 M),
71
3-
1
X
2 -
#,.♦•
• .. •
1 -
10
15
— T"
20
Figure 24. Lineweaver-Burk Transfomal-icn of Stearate Uptake Data.
The initial rates of uptake for 4 concentrations of stearate
were plotted by the double reciprocal method. Values for
velocities and concentrations are the same as for Fjgure 23.
The rate for S = 4.2 x 10"'' M was onitted.
72
for palraitate and 0.60 x 10 for stearatc. At naturally occurring
c?]icentrations the oysters are able to remove both palmitate and stearate
from the water because palmitate is below the lialf-saturating concentration
and stearate is about equal to the half-saturating concentration. Other
data on the fatty acid distribution indicate that the levels of palm.itate
may represent a greater percentage of the total free fatty acid and
stearate a lower percentage for other areas and methods of deternlnation
(Jeffrey, 1970). Our evidence then indiciited that the aiiimais had a
system which is saturated at 10 M which enables them to remove palmitate
and stearate at naturally occurring concentrations.
Uptake measurements were made with oleic acid at a range of concen-
trations from 1.25 - 15.0 x 10 N- The initial rates of the uptal.e are
shovm in the com.puter plot of least squares ve].ocities in Figure 25.
The velocities are only linear for the first 30 to ^.5 minutes and show a
saturation at longer times. I'Jhen the initial rates of uptake are plotted,
-6
a linear relationship is found with no saturation even at a 1.5 x 10 M
concentration. (See Figure 26.) The ambient concentration of oleate
-9
xn the sea water at Shell Mound was determined to be 0.7 x 10 M. At
this concentration, much less than those used in the uptake experiments,
the rate of uptake is essentially zero. Froi.T these data the uptake of
oleate from naturally occurring concentrations is not. significant and
represents a very small contribution to the total fatty acid removed from
sea water.
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77
Lipids of Cvassostvea and the
Incorporation of Labeled Fatty Acids
The neutral lipids of Cvassostvea vivginica have been characterized
by column chromatography and thin layer chromatography (Watanabe and Aclanan,
1972). We , found 5 major classes of neutral lipids as can be seen from a
14
TLC of the lipid extracts from a [ C]palmitate incorporation experiment
in Figure 27. The classes listed in order of increasing Rf are sterols,
triglycerides, alkyl diglycerides, wax esters, and cholesterol esters.
The polar lipids, which remain at the origin in a neutral lipid TLC
system, can be separated in a polar solvent system as described in the
methods section. In the lipid extract of oysters there are 4 or 5 major
polar lipid classes as can be seen from a TLC from a palraitate uptake
experiment in Figure 28. The 2 major compounds are those with relative
mobilities of 0.3 and 0.63, phosphatidyl choline and phosphatidyl
ethanolamine, respectively.
The genus Cvassostvea^ unlike the genus Ostvea^ contains no free
fatty acid pools in the lipid extracts (Watanabe and Ackman, 1972). This
fact is most important in evaluation of uptake experiments since any free
fatty acid that is assimilated is either incorporated into an esterified
lipid or catabolized for energy. Also, there is no problein of back
diffusion of a labeled acid once it is incorporated into a large intra-
cellular pool, as is seen in amino acid uptake (Johannes et al. ^ 1569).
By determining the incorporation into specific lipids, the actual uptake
and incorporation rates can be measured and quant itated.
The radiochromatographic scans of the neutral and polar lipid
separated by TLC folJowin?; a 2.8 x 10 M palmitate uptake experiment are
shown in Figures 29 and 30. Superimposed on tlie scans are the traces of
-">
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Figure 27. The Thin Layer Chromatographic Separation of Oyster Neutral
Lipids.
The lipid extracts from a 2.8 x 10"'' M palmitate incorporation
experiments were run on the neutral lipid system parallel with
standard mixtures. The lipids were visualized with iodine.
(1 - 7): 200 pi of the lipid extracts for 0, 15, 30, 45, 60,
90, and 120 minute samples. (8): standard mixture containing
in order of increasing Rf : cholesterol, tripalmitin, 1 - slkyl
2, 3 dipalmitoyl diglyceride, hexadecyl palmitate, and choles-
teryl palmitate. (9) : standard mixture containing in order of
increasing Rf : polar lipids, cholesterol, free fatty acid,
triolein, methyl palmitate, and cholesterol oleate. The dotted
line at the top of the plate was the solvent front.
79
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Figure 28. The Thin Layer Chrovuc tographic Separation of Oyster Polar
Lipids.
The lipid extracts from a 2. '8 x 10"'^ palraitats incorporation
experiment were run on the polar lipid ILC system parallel
with stana.-.rd mixtures. The lipids were visualized with
iodine. (1 - 7): 200 jal of the lipid extracts for 0, 15, 30,
45, 60, 90, and 120 minute samples. (8) : standard of di-
myristyl phosphatidyl choline. (9) : standard mixture
containing in order of increasing Rf: lyso-phosphatidyl
choline, phospatidyl choline, phosphatidyl ethanolamine, and
cholesterol. The dotted line at the top was the solvent front,
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84
the lipids visualized by iodine vapor. In the neutral plate a large
14
aDiOunt of C activity was seen av, the origin, representing incorporation
into the pnospholipid niaterial. Incorporation was seen into triglycerides
and cholesterol. The label incorporated into cholesterol was shown to be
cholesterol and not phospholipid material by chromatography in a raore
polar solvent system in v/hich tlie sterols and the phospholipids V7ere n^ore
completely resolved. Very little incorporation v/as seen in the alkyl
dlglycerides and the cholesterol and wax esters.
The phospholipids were scraped from the origin of the neutral lipid
plate and run in the polar solvent system and scanned. The scan showed
2 major areas of incorporation at the positions corresponding to
phosphatidyl choline (Rf = 0.3) and phosphatidyl ethanolamine (Rf = 0.63).
If a two-dimensional plate was run in the solvents described in the
methods section, and all spots were removed and counted, only 2 areas
had any significant radioactivity: the areas corresponding to phospha-
tidyl choline and phosphatidyl etbanolaminf (''iee Figure 31).
The fatty acid distribution ia the esterified lipids was determined
for the total lipid extract and for the isolated triglycerides (Figures 32
and 33). The distribution indicated that palmitate was a major component
.of the esterified lipids in both the triglycerides and total lipid.
When the lipids were separated by TJC, and the individual compounds
which showed activity in the radiochroniatographic scaiis were counted and
quantitated, the typical pattern seen is shown in Figure 34. The major
lipids labeled were the phospholipids followed by the triglycerides and
cholesterol. Further characterization of the phospholipid in all experi-
ments indicated that over 90 percent of the activity was located in the
phosphatidyl choline with the remainder found in phosphatidyl ethanolamine.
85
Figure 31. The Two-di'iiensional TLC Separation of Oyster Phospholipids.
The 120 minute extract of a 2.8 x 10~'' M palmitate uptake
experiment was run in the tv;o dimensional solvent system
described in the methods. The separation achieved in the
first solvent system vas shown by the dotted outlines on the
left. The labeled materials were (A) standard phosphatidyl
choline run in the second solvent system, (B) phosphatidyl
choline in the oyster extract, and (C) phosphatidyl ethanola-
mine in the extract. The origin was spotted with 200 yi of
the chlorororm extract. The solvent fronts were shovTi by
the dotted line.
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92
Other neutral lipid classes were found to contain some C label in the
palmitate and stearate experiments, but the incorporation V7as not
significantly above the experimental background for counting and
quantitating techniques.
If the data for the concentration dependent incorporation into
phosphatidyl choline are plotted for the series of palmitate experiments,
the iiiitial velocities can be determined by the least squares computer
plot (see Figure 35). If the slopes are now plotted as dpm/yg incorpo-
rated/r.iin, a saturation plot is obtained (see Figure 36). The Lineweaver-
Burk treatment of these uptake-incorporation data, sho\'m in Figure 37,
indicates that the maximal rate of incorporation into the phosphatidyl
choline pool is 0.4 dpm/yg/min.
The uptake expressed in molar terms is 15 ymoles of fatty acid
incorporated into 1 mole of phosphatidyl choline per minute. The Km for
the incorporation, measured from the slope of the reciprocal plot is
3.3 X 10~^ M.
The value for the Km represents the combination of processes to
vhich it corresponds; it involves both an uptake event and an incorpora-
tion event which are quite distinct biochemically. The Km for the
14
incorporation of C label into total lipid, as measured before in
Figure 23, was 5.0 x 10 M, The difference between the values m.ay be
ascribed to the multiple biosynthetic events necessary to incorporate a
newly assimilated fatty acid into a phosphatidyl choline molecule. No
statement can be made concerning the absolute nature of these events, but
by using the data for incorporation of the fatty acdd into phospholipid
as a measure of uptake, the contribution of any back diffusion to the
uptake process becomes moot.
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95
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10 X Concentration (M)
Figure 36. The Concentration Dependent P^te of Incorporation of Palmitate
into Phosphatidyl Choline.
The initial rates of incorporation were determined from the
slopes of Figure 35 and plotted against the concentrations
■ of palmitate in the experiments.
96
V.
s
Figure 37. Lineweaver-Burk Transformation of Palmitate Incorporation
Data.
The initial rates of incorporation into phosphatidyl choline
for 4 concentrations of palmitate were plotted by the double
reciprocal method. The maximum velocity was determined by
the y-intercept and the Km for the process V7as determined
from the slope. (S = 10"'' M) (V = dpm/yg phosphatidyl choline/
min) .
97
The incorporation of stearate into total polar lipid pools is
treated in tlie same manner as the palraitate data. The plots of the
concentration dependence of uptake and the Lineweaver-Burk reciprocal
plot appear in Figures 38, 39, and 40. The maximal rate of incorporation
into total phospholipid js 3.4 ymoles stearate incorporated per 1 mole
of polar lipid per minute. The Km measured from the slope of the
reciprocal plot is 5. 9 x lo"^ M. The Km for the total uptake determined
from Figure 24 was 6.2 x lO"^ M. The Km for stearate incorporation into
phospholipid is, as it is for palmitate, a misleading number for it
represents both assimilation and incorporation.
Competitive Uptake
The investigations into amino acid and carbohydrate uptake by marine
invertebrates demonstrated specific inhibitions of such uptake by j-roups
of amino acids and metabolic analogs of carbohydrates. Testerman's (1972)
work on fatty acids revealed competition of oleic acid uptake by linoleic,
palmitic, and caproic acids. Our investigations on uptake by oysters
revealed that palraitate and stearate uptake was much greater than that of
oleate. The animals did not have a saturable tip take system for oleate;
therefore, the effect of naturally occurring concentrations of oleate
-9
(10 ) on the uptake of stearate was investigated. (See Table 7.)
Oleic acid in concentrations 10 times greater than that found in
sea water was shown to inhibit the uptake of stearic acid. The assimila-
tion of stearate or equimolar concentrations of oleate was completely
inhibited. The variability of the data in these competition experiments
using whole anim.als prevented determinations of the type of inhibition
and the inhibitor constants, but the data indicate that stearate uptake
can be inhibited by o]eic acid.
98
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5 .0
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2.0
1.0
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■A (C)
(A)
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O o ^' °
A o o » .
o o o
o »
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X
y°
Or o
o o
e
25
50
75
Time in Mxnutes
Figure 38. Concentration Dependent Incorporation of Stearate into Total
Phospholipids,
The increase in the specific activity of the tota.l phospho-
lipid fraction from the chloroform extracts was plotted
against time for 3 concentrations of stearate. The plots
were the same as described as in Figure 35. Stearate con-
centrations were (A) and X - 0.093 m, (B) and 0-0.14 pM,
and (C) and * - 0.28 yM. The specific activity for the
stearate isotope in all experiments was 14.0 yCi ^^C/ymole.
99
•H
E
60
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100.
c
o
a
jj
o
a
u
o
V
10 X Concentration (M)
Figure 39. The Concentration Dependent Rate of Incorporation of Stearate
into Total Phospholipid.
The initial rate of incorporation determined from the slopes
of Figure 38 V7as plotted against the concentrations of
•stearate in the experiments.
20
15
100
-•- •
' ..'
k
10
1?
16
1
F3.gure 40. Lineweaver-Burk' Transformation of Stearate Incorporation Data.
The initial rates of incorporation into phospholipid for
3 concentrations of palmitate were plotted by the double
reciprocal method. The maximum velocity was determined from
the y~intercept and the ICm for the process was determined
from the slope. (V = dpm/jjg total phospholioid/min) (S =
10-7 M).
101
Table 7. The Effect of Oleic Acid on Stciric Acid Uptake.
Stearate Oleate Rate of Stearate
Concentration Concentration Uptake^ in pmole/gr/hr
2.8 X 10~ M 0 0.91
2.8 X lO"'^ M 8.8 X 10~^ M 0.68
2.8 X lo""^ M 3.5 X lO"^ M 0.40
2.8 X 10~^ M 1.8 X lO"^ M 0.00
Determined from the least squares slope of initial velocity
measurements.
102
The effect of oleate on palmitate uptake was investigated at up
to 100 times the naturally occurring concentrations for oleate because no
effect of oleate at naturally occurring concentrations could be demon-
strated. The data in Table 8 indicate that a 1/1 molar ratio of oleate/
palmitate has little effect on the rate of assimilation, but a 2/1 ratio
Increases the rate of uptake of palmitate. The total concentrations of
the fatty acids in the last experiment (palmitate, oleate, and background
fatty acids in the sea water), now exceed the micellar concentration for
the solution and the mixed micellar aggregates are formed. The aggrega-
tion of these acids then promotes the uptake of the included palmitate
as was seen in the data for the uptake of large concentrations of palmi-
alone (Figure 21).
The concentration effects of added oleate in the palmitate uptake
experiments indicated the need for further work into this concept of
promoted uptake by particle generation. The effect o^' palmitate on the
oleate uptake was investigated and the results appear in Table 9. The
assimilation of oleate has been shown to be much less than palmitate
and stearate and not saturable at 10 M concentrations (Figure 26). If
palmitate is added to sea water containing 2.3 x 10~^ M oleate, the
rate of uptake of oleate increases. If a similar concentration of palmi-
tate is added to a 5.0 x 10~ M oleate solution, there is little effect
on the uptake. The results demonstrate that the addition of palmitate
promotes micellar aggregation and an increase in uptake; but the results
from the larger concentration may mean that there is a limit to this
effect on acids like oleate which are not trken up to any appreciable
extent by oysters.
In view of these d£i:.3 fro- the inhibition experiments the results
must be interpreted very c-refully. If a small inhibition is seen it
103
Table 8. The Effect of Oleic Acid on Palmitic Acid Uptake.
PalmJtate Oleate Rate of Palmitate
Concentration Concentration Uptake^ in pmole/gr/lir
2.8 X lO"'^ M 0 0.88
2.8 X 10~^ M 2.5 X lO"'' M 0.82
2.8 X lO"'' M 5.0 X 10~'^ M 1.20
Determined from the least squares slope of initial velocity
measurements.
104
Table 9. The Effect of Palmitic Acid on Oleic Acid Uptake,
Olcate Palmitate Rate of Oleate
Concentration Concentration Uptake^ in nmole/gr/hr
2.5 X 10~^ M 0 3.6
2.5 X lO"'' M 2.8 X lO"'' M 16.8
5.0 X lO"'^ M 0 13.5
5.0 y. 10~ M 2.8 X lO"^ M 15.9
Rates measured in least squares slope of initial velocity plots.
lOf
may ba entirely due to dilution effects. At large fatty acid concentra-
tions, an inhibition effect nay be masked by the promotion effects caused
by particulate formation. The data suggest an obvious inhibition of
stearate uptake by oleate, but no effect on palmitate uptake was seen
with oleate.
Turnover of Lipid Classes
Data on the rate of fatty acid incorporation into various lipid
classes have been obtained as described previously. In order to investi-
gate the extent of this incorporation and its importance to the lipid
metabolibia of the oyster, determinations were made of the lipid turnover
rate.
A method of lipid labeling with radioactive sodium acetate has
previously been applied in order to determine the relative metabolic
activities of various lipids in copepods (Farkas et at., 1973). This
method v^as applied to oysters by labeling for 18 hours with sodium
3
[ Hjacetate (8raCi) in artificial sea water. In these experiments the
assumptions are made that all lipid classes will be labeled within the
period of exposure and that the label will be incorporated in sufficient
amounts to make the specific activity determinations accurate. A pre-
liminary experiment with labeled acetate indicated that the acetate could
be removed from the sea water by the animals and that it was incorporated
into all the lipids of the chloroform extract.
Figure 41 shows the results of the labeling experiment in the loss
of label from the methanol and chloroform extracts cf the animals. The
incorporation is much gre^Ler in the non-lipid, methanol soluble material
indicating that the acetare has entered several metabolic pathways not
106
600
500 '
•H
&
60
e
(X
400- S
\
300 .
200
s.
e N.
(A)
i&
c
100 »
« %.
(B)
Time in Hours
Figure 41. The Turnover of Lipid and Non-lipid Compounds Labled with
[-^H]Acetate.
The animals were labeled for 18 hours in 4 liters of arti-
ficial sea water containing 8 m Ci-^H acetate at a
■ ■ concentration of 1. 3 x 10"-^ M. They were reT.cved and placed
in a non-labeled sea water medium. The radioactivity in the
methanol (A) and chloroform (B) extracts of oyster tissue was
plotted against time, after removal from the non-labeled sea
water.
107
leading to lipid synthesis, and that it has been metabolized to
labeled products which are themselves incorporated into methanol
extractable compounds.
The isolated lipid classes of triglycerides, total polar lipids,
and cholesterol were the only compounds with sufficient specific activity
to permit determinations of turnover rates. Figure 42 shows decrease in
the specific activity of each class versus time after the animals were
removed from the acetate labeled sea water and placed in unlabeled sea
water. There is a short lag of 60 minutes during which the maximum
incorporation occurs. This is due to the time required for the acetate
to enter the metabolic pools following its assimilation from the external
medium. The curve decreases in 5 hours to that turnover times may be
determined. The triglycerides are the most metabolically active lipid
class in the animal indicating that they represent the major energy
storage form in the oyster. The polar lipids are metabolically active
and important in the quantitative amounts which they represent, for up
to 60 percent of the lipid material in oysters is the polar lipid
fraction (Watanabe and Ackman, 1972). The large incorporation into the
phospholipid shown in Figure 34 may reflect both the turnover activity
and the large weight percentage that the phospholipids contribute. The
sterols and other neutral lipid compounds are not very active and have
a low rate of turnover.
ra
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CONCLUSIONS
The presence of amino acids and carbohydrates in sea water, and their
uptake by soft-bodied marine invertebrates have been demonstrated for at
least 5 different animal phyla (Stephens, 1964). The uptake of lipids,
specif ical.ly free fatty acids, has only been shown for 2 nereid species
(Testerman, 1972) and 2 pogonophoran species (Southward and Southv/ard ,
1972). The concentrations of free fatty acid used were 0.06 - 6.0 JiM
which approximated the range of concentrations of free fatty acid found
in the sea waters in v/hich the animals lived.
In tlie present work, we have demonstrated that the /jiierican oyster,
Crassostfea vLvg-tn'ioa, can remove palmitic and stearic acids from sea
water at concentrations as low as 0.0? pM. Tj;c naturally occurring
concentrations of lip:ids that we determined for the sea water from the
Shell Kound estuary were 280 pg/liter total lipid including up to
77 yg/ldtet of total free fatty acid (equivalent to a 0.3 yM solution of
palmitate) .
The uptake of palmitic acid was saD.-m to be conzpletely inhibited by
200 mM sodium cyanide, indicating an energy dependent step in the
process. V'e have shown that the loss of labeled palmitate from sea water
is physiological and due, only in a small part, to chemical adsorption
of the fatty acid onto the shell of the animals -..I'd glass walls of the
experimental apparatus. The loss in label from the sea water occurred
rapidly, within the first 60 - 90 minutes, and was concurrent with the
appearance of radioactivity in the animal extracts.
T
110
Ill
The concentration dependent uptake experiments revealed that
palmitate and stearate are assimilated by saturable uptake systems, but
oleate is not. At concentrations above the saturated level (0.5 -
0.6 ]M) , the uptake of palmitate and stearate abruptly increases. This
increased uptake may be due to self-aggregation of the fatty acid
molecules into large raicellar particles which are then filterable by the
oysters. Fatty acids in sea water at concentrations in the range of
0.1 pM vnMl occur in the form of small molecular aggregates since lipids
are hydrophobic and have natural tendencies to aggregate in aqueous
media; but these aggregates are too small to be filterable by the oysters'
normal filter-feeding apparatus. At artificially increased concentra-
tions (0.5 - 0.6 ).iM) , these molecular aggregates increase in size and
become greater than 0. 5 \im approaching the lower size limit for the
oysters' cilir.ry-mucoid filtration system, thus increasing the uptalic.
We have shown that the uptake of radioactively labeled celite
particles of sufficient size, 50 ym, to be filtered by the oysters' filter-
feeding system differs from the uptake of freely soluble fatty acid in
the time sequence involved. Soluble fatty acid can begin to accumulate
in the lipid pools during the initial 15 minutes of exposure, but the
celite filtration requires more than 30 minutes before incorporation is
seen. This observation, along with our findings on the tiT-ae course of
uptake of celite containing adsorbed aniline dye, confirms the auto-
radiographic observations made on C amino acid uptake by another
lamellibranch species (Pequignat, 1973).
Our results with the temperature dependence of the uptake process
indicated a depressed uptake rate st intermediate temperatures and a
totilly negative uptake at 20 C, a temperature to which the animal vrould
112
be exposed environmentally. These results may indicate a physiolojical
reaction of the animals to temperature rather than a metabolic one. In
shelled animals, such as oysters, v.'hich can seal themselves off from their
milieu, the investigation of processes requiring exposure of the animal
to tlie media is dependent upon the physiological stimuli to which the
animal normally responds.
The experiments on the inhibition of uptake by competing fatty
acids revealed that stearate uptake can be inhibited by low concentrations
of oleate. Investigations into the effect of oleate upon palmitate uptake
showed no inhibition up to a l/l oleate/palmitate molar ratio, but at a
2/1 ratio, the uptake of palmitate was promoted. We showed that the rate
of oleic acid uptake was very small in comparison to that of palmitate and
stearate, tut that by adding unlabeled palmitate to labeled oleate, the
rate of uptake of oleate could be increased. The results seem zo
indicate, once again, the recurring observation that the rate of uptake
of dissolved m.ate.rial can occur in the absence of filtration feeding,
but that when a concentration dependent micellar aggregation occurs, an
increase ir the assimilation rate due to filtration feeding is seen.
The results of any inhibition studies at elevated concentrations should,
theielore, be interpreted carefully; inhibition of the uptake systems for
dissolved lipids may be masked by the promotion effects due to particle
formation.
The results of the incorporation experim.ents show that palmitate
and stearate are major fatty acids in the esterified lipids of the oyster.
The labeled fatty acid rem.oved from the se i water by the animal is
e3terified immediately into the complex lipids, for the animal does not
have a large free fatty acid pool. The fatty acid is incorporated into
113
all the lipid classes, but the major incorporation occurs into the phospho-
lipids, primarily phosphatidyl choline, and into the triglycerides. The
presence of label in the cholesterol fraction indicates that the animals
were viable and metabolically active for the fatty acid must be broken
do;m to acetate before steroid synthesis can occur. The levels of incorpo-
ration into the triglycerides varied from one experiment, and even from one
group of animals, to the next. The large turnover rate seen for the tri-
glycerides helps to explain thi| variation; the triglycerides are the major
lipid energy storage form in the oyster. Therefore, the concentrations of
triglycerides would depend upon the length of time the animals had been
without adequate food. In negative energy debt, the fatty acids being
assimilated would be used for energy and not the synthesis of a storage
form.
The importance of the uptake of freely dissolved lipid in the form
of fatty acids for the energetic needs of the animal can be determined
from the maximum velicity of uptake. Ue found in open shell experir.ents
with palmitate that 0.26 ymoles of fatty acid are lost from the sea water
in 2 hours and that 0.147 ymoles are taken up into the lipid extracts of
the animals. This uptai;e represented incorporation of the palmitate
removed into esterifieo lipid, since no free fatty acid was found in the
lipid extracts. A small amount of the label lost in the experin.ent is lost
due tc adsorption onto the glass surfaces and the shells of the animals,
but the majority is lost due to adsorption onto tne feces and pseudofeces
of the animals and onto the surface of the water itself. The uptake into
the chloroform extracts of the animal and th- small amount of non-lipid
incorporation seen in the methancl extracts account for over 50 percent of
the label lost from the sea watsr during the experiments. If the maximum
rate of uptake is 2.30 pmoles/gr/hr , as m^easured from our experiments, and
114
the average oyster weight is taken as 3.5 grams, then the uptake rate per
oystt-r per hours would be 8.05 pmoles/aniioal/hr . If this is converted to
weight/animal/hr for palmitic acid, the rate v.'ould be 2.1 yg/gr/hr. This
is small relative to the 0.16 mg carbon/hr that an oyster normally-
removes froi'i the sea v/ater for its metabolic needs (Nicol, 1970), but
when one considers all the lipid available to the animal, the accelerated
rate when particulate matter is formed, and the range of concentrations
found in natural waters, this pathway becomes more important energetically.
An important implication of a free fatty acid uptake system is in
the physical sim.ilarity of the fatty acid and other lipid material to the
hydrocarbon pollutants found in our coastal waters. Oysters are known to
concentrate petro-hydrocarbons (Stegeman and Teal, 1973) from sea water
and store them for several months. Very few metabolic interconversions
occur during this time and it appears that the petro-hydrocarbons are
merely dissolved in Che lipid pools of the animal. Trie uptake of these
compounds must occur by a pathway similar to that utilized for free lipid
uptake. Long after an oil slick on the surface has dissipated, the
animals can still remove hydrophobic material dissolved in sea water.
The latest research into the iyt vivo and in vitro uptake of dissolved
organics by lamellibranch molluscs (Bamford and KcCrea, 1975) indicates
, that these animals may remove a certain percentage of particulate-adsorbed
organic material by extra-brachial en^:yme secretion, breakdown, and uptake
directly acorss the gill surface, rather than ciliary transport of the
particles to the mouth. Future work on the mechanisms of uptake of
esterified materials is certainly indicated.
Work done by Ryther and his colleagues at Woods Hole Marine Biological
Laboratory has shovm that the American oyster, C/'assostrea vivginica, is a good
candidate for exploitation by aquaculture technology (Ryther et dl. ^ 1972;
115
Tenore et al. , 1973). In their work with tertiary treatment of municipal
sewage by algal farming, the oyster was used as a primary consumer of
algal Material grown in diluted sewage effluent. The oysters grew to
full harvestable size in a matter of nine months on this algal diet
(Ryther et al. , 1972). The apparent efficiency of the animal in converting
nutrients to body mass may be due in a large part to direct uptake path-
ways involving the elevated concentrations of dissolved nutrients that
would be in the sewage effluent, which may not be completely utilized by
the algal cultures. This pathway of direct assimilation of lipid
material which we have demonstrated may be very important to the future
farming of animals in our coastal waters.
The appearance of such an assimilatory pathway in marine invertebrates
has been demonstrated. The presence of dissolved organic material In
fresh and brackir.h waters and its utilisation by fresh water lame] libianchs
should be investigated, for it may reveal information on the universality
of these processes in all soft-bodied aquatic invertebrates.
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BIOGRAPHIC.\L SKETCH
Terry Allan Bunde was born on January 11, 1947, in Orlando,
Florida. He was raised in Orlando and following his Graduation from
high school he entered Rollins College, Winter Park, Florida where he
majored in pre-medical science. He received his Bachelor of Science
degree in 1968.
He entered the Department of Biochemistry in the graduate school
at the University of Florida in 1968 and worked toward his degree until
he was drafted in 1969. After two years in the United States Army, he
reentered the Department of Biochemistry in the graduate school of the
University of Florida in September, 1971. Since then, he has pursued
his work toward the degree of Doctor of Philosophy in the Biochemistry
Department,
He was married to the former Pamela Sue Riess in August, 2 971.
1^:0
1 certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Melvin Fried, Chairman
Professor of Biochemistry
1 certify that I have rr^-^d this study and that in my opinion it
conforms to acceptable standaris of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Pliilosophy.
Cz^.j^, ,^/ /^i6^ ^.
Charles M. Allen, Jr.
Associate Professor of Biochemistry
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adf-quate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
-U
Samuel Gurin
Professor of Biochemistry
.L certify that I have read this study ard that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, r.^- a dissertation for the degree of
Doctor of Fhilosophy.
■ — '-"7
William E. Carr
Associate Professor. Zoology
Ihis dissertation was submitted to the Graduate Faculty of the
Department of Bxcchen^istry in the College of .Arts and Sciences and to
the Graduate Council, and was accepted as partial fulfiJlment of thl
requirements for the degree of Doctor of Philosophy.'
June, 1975
Dean, Graduate School
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