Citation
The Uptake of free fatty acids from sea water by a marine filter feeder, Crassostrea virginica

Material Information

Title:
The Uptake of free fatty acids from sea water by a marine filter feeder, Crassostrea virginica
Creator:
Bunde, Terry Allen, 1947- ( Dissertant )
Fried, Melvin ( Thesis advisor )
Allen, Charles M. ( Reviewer )
Gurin, Samuel ( Reviewer )
Carr, William ( Reviewer )
Place of Publication:
Gainesville, Fla.
Publisher:
University of Florida
Publication Date:
Copyright Date:
1975
Language:
English
Physical Description:
ix, 120 leaves : ill. ; 28cm.

Subjects

Subjects / Keywords:
Amino acids ( jstor )
Animals ( jstor )
Eggshells ( jstor )
Fatty acids ( jstor )
Lipids ( jstor )
Nonesterified fatty acids ( jstor )
Oysters ( jstor )
Palmitates ( jstor )
Sea water ( jstor )
Stearates ( jstor )
Biochemistry and Molecular Biology thesis Ph. D ( local )
Dissertations, Academic -- Biochemistry and Molecular Biology -- UF ( LOCAL )
Fatty acids ( lcsh )
Oysters ( lcsh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Abstract:
The ability of the American oyster, Crassostrea virginica to remove naturally occurring dissolved free fatty acids, inconcentrations approximating those found in sea water, was investigated using radioactive isotopes of palmitate, stearatc, and olcate. Petroleum ether (30 - 60°C) extracts of the sea water from a Florida Gulf Coast estuary contained up to 280 μg of total lipid material per liter including 77 yg of free fatty acid. Thefatty acids, separated by gas liquid chromatography, were predominatly saturated with even carbon numbers. The major fatty acid present was palmitate. The animals were shown to remove labeled palmitate from sea water by measuring the appearance of the radio-activity iu the chloroform extractable material. The uptake process was shown to be physiological and not chemical adsorption onto shells. This assimilation was Inhibited ,7±za 200 ml-I sodium cyanide. The temperatcre 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 and palmitate demonstrated that the process 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 concentration of dissolved material. The kinetics of the uptake into chloroform extractable material was investigated for palnitate, stearate, and oleate. Both palpitate and stearate shoved saturable uptake systems as determined from reciprocal rate-concentration plots. The rate of uptake of both acids markedly increased when nicellar concentrations of the fatty acids were reached. The rate of uptake of oleate was much less than that for palmitate and stearate, and was not saturable at natural concentrations. The rate of uptake into isolatable lipid classes was investigated; the major species labeled were phosphatidyl choline, triglycerides, and cholesterol. The rates of incorporatron of palmitate into 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 with sodium. [^'acetate, removing the label, and following the decrease in specific activity of each lipid with time. The contribution of the uptake process to the total metabolic needs of the animal was estimated. The impact of such lipid uptake studies was discussed in light of municipal sewage and petrochemical pollution of natural oyster habitats as well as the selection of oysters as a possible aquaculture species.
Thesis:
Thesis--University of Florida.
Bibliography:
Bibliography: leaves 116-119.
Additional Physical Form:
Also available on World Wide Web
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Terry Allen Bunde.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
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1'. ,,



TERRY ALLEN BULDE


















. :1. : 'I 7 - ,'L'-i.. -













A'INOLEDGE, FT-TS


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 thanks are given to the author's fellow graduate students

for the suggestions and encouragement they offered. Thanks are also

offrr d especIllyl to Mr. William Gilbert for hl assistance in the

preparation of the 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 long hours

of typing, this work would never have been completed.














TABLE OF CONTENTS


Page
ACKNOWLEDGEMENTS.. . . . . . . . . . . ii

LIST OF TABLES . . . . . . . . .. . . . . iv

LIST OF FIGURES . . . . . . . . .. . . . .v

ABSTRACT . . . . . . . .. . . . .. v. iii

INTRODUCTION . . . . . . . . .. . . . . .1

Metabolic Significance of Dissolved Organic Matter .. 1
Lipids and Free Fatty Acids in the Marine Food Chain 11
The Oyster as a Possible Experimental Subject for Lipid
Uptake . . . . . . . . . . . .14
Research Objectives . . . . . . . . .. . 16

MATETRIATS AND METHODS .. .... . . . . . . .. 17

Materials . . . . . . . . ... . . . 17
Methods . . . ..... . . . . . 20

DATA AND DISCUSSION . . . . . . . . ... . . 31

Lipids and Free Fatty Acids in Sea Water . . . .. 31
Uptake of Palmitic Acid . . . . . . . . . 39
Celite Uptake Experiments . . . . . . . ... 50
Concentratior Dependent Uptake--Kinetic-Parameters of
Uptake . . . . . . . . .. . . . .60
Lipids of Crassostrea and the Incorporation of Labeled
Fatty Acids, . . . . . . . . ... . 77
Competitive Uptake . . . . . . . . .. . 97
Turnover of Lipid Classes . . . . . . . .. 105

CONCLUSIONS . . . . . . . . . . . . . 110

BIBL0CPOGAPHY .......................... 116

IOG.RAP7ICAL SKETCH . . . . . . . . . 120













LIST OF TABLES


Table Page

1 Amino Acids and Glucose Uptake . . . . . .

2 Fatty Acias in Marine Waters . . . . . . ... 32

3 Visualization Reagents for TLC . . . . . . .. 27

4 Concentrations of Exrractable Specific Lipids in the Sea
Water Collected onr June 21, 1.974 (Extract A) and
March 31, 1975 (Extract B). . . . . . . 3

5 The Free Fatty Acids in the June 21 Sea Water
Extraction . . . . . . . . ... . 38

6 Localization ot Oil Red O Celite ParticJes Removed fruo~
Sea Water by Experimental Animals .. . . .... 54

7 The Effect of Oleic Acid on Stetric Acid Ulptake .... 101

8 The Effect of Oleic Acid on Panlmitic Acid Uptake ..... 103

9 The Effect of Palmitic Acid on Olcic Acid Uptake . .. 104













LIST OF FIGURES

Figure Page

1 The Flow of Organic Compounds in the Marine Ecosystem 3

2 Cycling of Organic Matter by the Benthos . . . . 4

3 Extraction with Adapted Bloor Method . . . ... 21

4 Extraction with Adapted Bligh and Dyer Method ..... 22

5 Separation of Polar Lipids in Sea Water Extracts . . 32

6 Separation of Neutral Lipids in Sea Water Extracts . 33

7 Gas Liquid Chromatograph of Fatty Acid Methyl Esters
Prepared from Sea Water Extract of June 21 .... 37

8 Diffusion of Adsorbed Labeled Fatty Acid into a Sea Water
Wash Saturated with Unlabeled Palmitate . . .. 40

9 The Uptake of Palmitic Acid Measured Using the Bloor
Extraction Technique . . . . . . .... 43

14
10 Removal of C Fatty Acid by Background Adsorption onto
Shells and Glass Surfaces . . . . .... 45

11 The Uptake of Palmitic Acid in the Presence of 200 mM
Sodium Cyanide . . . . . . . . ... .47

12 The Uptake of Palmitate, Double Addition of Label . .. 49

13 The Uptake of Palmitic Acid by Open-Shell Animals . .. 51

14 Temperature Dependent Uptake of Palmitate . . . . 52

15 The Uptake of Celite-adsorbed Palmitate . . . .. 56

16 The Uptake of Celite-adsorbed Palmitate, Open Shell
Animals . . . . . . .. . .. . ... 57
-7
17 The Uptake of 2.8 x 107 M Palmitate, Celite-adsorbed
and Free . . . . . . . . ... . . 59
-7
18 The Uptake of 2.8 x 10- M Stearate, Celite-adsorbed
and Free . . . . . . . . ... . . 62






LIST OF FIGURES--ContJnued


Figure Page

19 Concentration Dependent Uptake of Palmitate .... .64

20 Concentration Dependent Uptake of Stearate .. . 66

21 The Concentration Dependent Pate of Uptake of
Palmitate . . . . . . . . . . 67

22 The Concentration Dependent Rate of Uptake of
Stearate . . . . . . . . . . 63

23 Lineweaver-Burk Transformation of Palnitate Uptake
Data . . . . . . . . . . . 70

24 Lineweaver-Burk Transformation of Stearate Uptake
Data . . . . . . . . . . . 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 . . . . . . . . . 78

28 The Thin Layer Chromatographic Separaticnl of Oyster
Polar Lipids . . . . . . . . . 79

29 Radiochromatographic Scan of the Neutral Lipid TLC
Separation . . . . . . . . . . 81

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
Prepc.red from Esterificd Fatty Acids of Isolated
Oyster Triglycerides . . . . .. . .7

33 Gas LiqJid Chromatg-raph of Fatty Acid Methyl Esters
Prepared fro-i Elterified Fatty Acids of Oyster
Total Lipid l.-:r-acts . .. . . . . . 89

34 Incorporation of C labeled Palr itate into Isolated
Lipid Classes . . . . . . . .. 91

35 Concentration Dependent In'"oruratioi. of Palmitate into
Phosphatid)l Choliie . .. ...... 94







LIST OF FIGURES--Continued


Figure Page

36 The Concentration Dependent Rate of Incorporation of
Palmitate into Phosphatidyl Choline ..... .95

37 Lineweaver-Burk Transformation of Palmitate Incorpora-
tion Data . . . . . . . ... . .96

38 Concentration Dependent Incorporation of Stearate
into Total Phospholipids . . . . . . 98

39 The Concentration Dependent Rate of Incorporation of
Stearate into Total Phospholipid . . ... .99

40 Lineweaver-Burk TransformaLion of Stearate Incorpora-
tion Data . . . . . . . .... .. 100

41 The Turnover of Lipid and Non-lipid Compounds Labeled
with [3H]Acetate . . . . . . ... 106

42 The Turnover of Specific Lipid Classes in the
Chloroform Extracts of Oysters Labeled with
[3HJAcetate . . . . . . . . . 109





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, CRASSOSTRE1A VIRICI.ICA

By

Terry Allan Bunde

June, 1975


Chairman: Melvin Fried
Major Department: Biochemistry


The ability of the American oyster, Crassoasrrea virvinica too

remove naturally occurring dissolved free fatty acids, in concentrations

approximating those found in sea water, was i,-vestigated usiog radiicUtive

isotopes of palmitate, stearate, and oleate.

Fceroicum ether (30 GO C) extracts of the sea water from a fior.da

Gulf Coast estuary contained up to 230 pg of total lipid material pcr

liter including 77 pg of free fatty acid. Th: fatty acids, separated by

gas liquid chromatography, were predominatcl saturated with even carbon

numbers. The major fatty acid p-:csent was palmitate.

The animals were shown to remove labeled palmitate from sea water by

measuring the appearance of the radioictivity in the chloroform extract-

able material. The ipt.e process was shown to be physiological :-d r.iA;

chemical adsorption orntc shells. This assimilation was inhibited with

200 m1M sodium cyanide, The temperatLre dependence of the uptake process

was investigated at 20, 25, 30, and 35C.

The rate of uptake of 50 ipm cej ite particles carrying adsorbed

radioactively labeled stearate and palmitate demonstrated that the process


viii






of filtration feeding was not responsible for the removal of freely

dissolved fatty acids. The rate of uptake of celite bound material was

delayed by 30 minutes when compared to the uptake of an equal concentra-

tion of dissolved material.

The kinetics of the uptake into chloroform extractable material was

investigated for palmitate, stearate, and oleate. Both palmitate and

stearate showed saturable uptake systems as determined 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 much less than that for palmitate and

stearate, and was not saturable at natural concentrations.

The rate of uptake into isolatable lipid classes was investigated;

the major species labeled were phosphatidyl choline, triglycerides, and

cholesterol. The rates of incorporation of palmita.e into phosphaLidyl

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

with sodium ["Hlacetate, removing the label, and following the decrease

in specific activity of each lipid with time.

The contribution of the uptake process to the total metabolic needs

of the animal was estimated. The impact of such lipid uptake studies was

discussed in light of municipal sewage and petrochemical pollution of

natural oyster habitats as well as the selection of oysters as a possible

aquaculture species.












INTRODUCTION


Metabolic Significance of Dissolved Organic Matter


The salt waters of the world contain relatively constant concentra-

tions of inorganic compounds, evidencing only small changes in salinJiy,

but they show orders of magnitude variation with tire and location in

concentrations of dissolved organic matter and dissolved particulate

matter (Wagner, 1969; Duursma, 1961). Early investigations of dissolved

organic substances were hampered by crude ucthods of sampling, analysis,

and quantitation; but with newer, moe' refined techniques, it has become

apparent that the oceans of the world contain more dissolved organic

matter than that which is represented by the entire living bio.asc 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 lipids. These

materials share the common property of being able to pass through a

0.45 pm cellulose acetate filter and are, therefore, distinguishable from

the particulate matter vhich 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 ecOn:3stem,

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--exchange, solvent-extraction,







and co-precipitation (Wagner, 1969). The difficulties inherent in measuring

mg/liter or pg/liter quantities of organic compounds in solutions containing

g/liter quantities of inorganic salts have made quantitation difficult, but

reliable data show cotal amino acid concentrations of 30 pg/liter of which

16 pg/liter is glycine (Hobbie et aZ., 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 interrelationships 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 Duursma (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 result in these molecules. The primary producers in a salt water

ecosystem, the phytoplankt.r"' have been shown to lose a large amount of

their photosynthetic products through leakage and overproduction, up to

1-40 mg Carbon/m3 sea water/day depending upon the water depth and latitude

(Thomas, 1971). The zooplankters which consume the primary producers, a.so

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 matter by

the be-nthic animal communities are as complex and as important as t-c Lio-

rbe.ical interconversions that occur. The pools of detritus and dissolved

organic matter are net static but in a constant dynamic state as ;ae .,e

organisms at eacii trophic level.






Ligh t
*i

___ Assimi aLion
Living Organic
Matter Excretion
---------r--

SFilter Dying and
Feeding \ xcr.tion





Assimilation
\of DisF;olved
Organic Matter

Excretion and Detritus
Overproduction \


Rain Atmosphere

E

NO3 CO
3 2
NO. I1 CO
2 3
-3
PO3 HCO

3 3
NH3 CO3
-- - -i


/7


Mineralization


/






/







Bacteria]
interconversions


- -- Fresh Water Runoff


Figure 1. The Flow of Organic Compounds in the Marine Ecosystem.
Taken from: Duursma, E.K. Netherlands J. of Sea Res. 1:4
J(1961).








ALLOCHTHONOUS SOURCES


Macroscopi
and Assort
Epibiota







AUTOCHTHONC
SOURCES


c Plants Phytoplankton, Organic
ed Zooplankton, and Precipitates
Their Feces I






BENTHOS ----


)US


/
/
/
/


Terrestrial
Organic lMatter








Microflora and
Protozoans


Dissolved _
Organic ---BACTERIA
Organic 4-------- BACTERI:A


Matter


Feces and Pseudofeces
Foc A oreO atsC


Encrusted Mineral




Respiration Export



Accumulation
in Sediments


Grains


Figure 2. Cycling of Organic Matter by the Benthos.

This figure taken from Johnson (1974) depicts the function
of the benthos or bottom dwelling organisms in the processing
of organic matter. The external sources of organic matter
are shown at the top and the sinks at the bottom. The
bacteria are the major organisms involved in processing the
material into freely dissolved matter.

Taken from: Johnson, R.G. J. Mar. Res. 32(2):326 (1974).


I




5



In 1908 a German biologist, Putter, 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 (Putter, 1908). His theory was considered valid until Krogh

(1931) showed that Putter'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 (1946, 1949, and 1961), whose views of utilization 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 phosphurous 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.

With the presence of dissolved organic matter in sea water an

undisputed fact, research was initiated into elucidating the physical and

chemical forces that convert these organic compounds into particulate

matter of sufficient size for filtration or adsorption methods to be

used by marine organisms in their removal from the sea water. The initial

studies employed fish hemoglobin as a substrata for coalescence of organic

matter from sea water. This uiaterial was then shown to be important in







the nutrition of invertebrates in feeding experiments (Fox et a/c., 1953).

Studies into the geophysical forces involved in those sea surface currents

known as Langmuir circulation and the production of foam lines at their

interface led researchers to formulate the thesis that organic partic'j-

lates formed at disturbed air-water interfaces (Baylor et aZ., 1962;

Riley. 1963; Sutcliffe et al., 1963). The formulation of organo-phosphate

containing particles at such disturbed surfaces, and the resultant accumu-

lation of phosphate containing material when these particles were isolated

and added to 0.45 pm filtered sea water are examples of the process

whereby these particles may be formed and increase in size (Sutcliffe et al.,

1963).

laylor and Sutcliffe isolated organic particulate matter from

despurated sea water following filtration through a 0.45 uIm filter and

demonstrated the survival of Artcmia cultures fed this matr-iil for

J.1-16 days (Eaylor et al., 1963). The cultures survived and grew as well

&as those fed yeast extracts. The data of these investigators seemed to

support Piutter's original ideas and provided an impetus for further work

based on the hypothesis that dissolved organic were metabolically 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

ereas 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 acids and carbohydrates, at naturally occurring con-

centrations, were removed from sea water solutions by several marine

species (see Table 1). Based on results with radioactive tracers, the








Table 1. Amino Acids and Glucose Uptake.


SOrganic m Concentration
Author Date Organism
Compound Tested


Collier
et al.


Stephens. b
Schinskec

Stephens &
Schinske

Stephens


Stephens


Stephens


Stephens 6
Virkan


Reish &
Stephens

Anderson &
Stepihens


Taylor



Chier
eit al.


1953 Glucose


1958 Amino Acid
(Glycine)

1961 Amino Acid
(Glycine)

1962 Glucose


1963 Amino Acids
(Ala, Gly)

1964 Amino Acid
(Glycine)


1966 Amino Acids



1969 Amino Acid
(Glycine)

1969 Amino Acid
(Glycine)

1969 Glucose and
Amino Acids


1972 Amino Acids


Shick 1973 Amino Acid
(Glycine)


Crassostrea
virginica


12 invertebrate
phyla

11 invertebrate
phyla


Fungia
scutaria


Clynenel la
torquata

Nereis
Zirrnnicola
and succinea

Ophiactis
arenostc


Neanthes
areraccodentava


Crustaceans


Ner .eis
oire-ns
and sars

Glycere


.4ure lia
aurita


1 x 10-3

-'3
2 x 10


2 x 10-3
2x i.0


-5
4.7 x 105

-6
1 x 10-

-5
2 x 105



2 x 10-8

3 x 10 -

1 x 10-7


4.3 x 10-7


2 x 10-5


-6
1 x 106


1.27 x 10-6
1.27 x ]0








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: coelenterates, annelids,

crustaceans, molluscs, and echinoderms, and showed that at least some

capacity to remove dissolved free amino acids or carbohydrates exists in

all 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

was determined.

Fergi son, using an autoradiographic technique Lo study the uptake of

amino 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 pseudopods, were later transported

throughout the water vascular system. The uptake was quantitated by

counting silver grains in the photographic emulsions (Ferguson, 1970,

.1971). Other work with starfish species (Pequignat. 1972) demonstrated

amino acid uptake into an isolated arm of i enricia sanquinolenta by

autoradiographic techniques. The labeled amino acid, as in Ferguson's

studies, could be seen to be incorporated almost exclusively into the

ambulacra and aboral wai. of the arm placed in the sea water. Time

course studies revealed major incorporation of the amino acid into

proteins of the gonadal tissue, indicating significant i.tilization of

this dissolved material assimilate from sea water.







It is apparent from these studies that animals with 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 are filter-feeders, starfish and

urchins are herbivores, and coelenterates are carnivores, but a3l 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 acid uptake

from ambient sea water concentrations of 10- 10 M was shown, 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, 3971, 1972). Uptake of such compounds by the pogonophorans

apparently differs only from that in the polychaetes (Taylor, 1969; Stephens,

3964) in that the efficiency with which pogonophorans remove dissolved

substances is much better, i.e., they are better adapted to environments

wherein concentrations of amino acids and fatty acids are less than 10-5 M

(Southward and Southward, 1971). The studies of these animals indicate that

as much as 50 percent of their metabolic needs can be met by the dissolved

organic in the sea water around them. 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 optimum usage of such pools

can be maintained.








The basic in vivo experimental techniques of Stephens and indeed

of all others who have looked at uptake of dissolved material from sea

water, i.e., the use of tracer methods to yield some indication of the

percentage of the metabolic needs met by these substances, have been

challenged by Johannes et al. (1969). In experiments with the marine

turbellarian Bdelloura, these workers found that this animal leaked amino

acids into the medium at a faster rate than it removed 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 that when a section of body wall of the blood worm Glycera was

removed and placed in a Ussing chamber in which the flux of amino acids

into and out of the organism could be measured, the net flux was into the

animal.

The metabolic significance of the work with amino acid uptake is

complicated by the function of the molecules as ocnoregulators 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 C'lymeneila torquata

and showed that at low NaCI concentrations the uptake was virtually zero.

At these salt concentrations this animal would be actively lowering its

internal pool of amino acids to compensate for decreased ionic concentra-

tion in the medium.

The ability to remove dissolved amino acids and carbohydrates at

isoionic sea water concentrations, however, is real and their net movement








into the organism may be important 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 pm 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 Southward (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 are exposed in their natural

environment. The fatty acids, once removed, were incorporated into

several complex lipid compounds. The loss of label from these organisms








Table 2. Fatty Acids in Marine Waters.


Method of Concentration Invest
Compound in mInvestigator(s)
Extraction in mg/1


Fatty Acids



Fatty Acids


Lipids


Fatty Alcohols,
Acids, Esters,
and HC


Fatty Acids


Lipids/Fatty
Acids


Liquid-Liquid//
pH 3//ethyl acetate


Liquid-Liquid//
CC14 + CHCl3


Liquid-Liquid//
pH 2//petroleum
ether + ethyl
acetate

Coprecipitation w/
FeCl //extract w/
CHC13


Liquid-Liquid//
pH 2.0-2.5//
Extract w/CHC1
-3

Liquid-Liquid//
pH 2//CHC1
(saponfication)


0.1 0.8



0.01 0.12



0.4 8.0


0.2 1.0


0.01 0.025


0.11 0.13
0.06 0.05


Slowey
et al.,
1959, 1962

Williams,
1961, 1965


Jeffrey,
1962, 1966


Garrett,
1967


Stauffer &
Macintyre
19 970


Testerman,
1972


Source: Taken in part from Jeffrey (1970) and Testerman (19721.








into the medium, the so-called "leakage" rate, was only 5 percent, with

the majority of the "leaked" radioactivity being in the form uf 14CO,

indicating the catabolism of the free fatty acid (Testerman, 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 Crassostrea virginica and Mytilus edulis

were able to remove significant quantities of sub-lethal concentrations of

petro-lipid material, up to 50 ug/gram wet body weight. This lipid material

was assimilated in the gill and mantJe areas as well as in the gut, indicating

a possible direct adsorption pathway (Lee et al., 1972). The naturally

occurring hydrocarbons in the lipid poc2s of the organisms were not as

saturated nor as aromatic in nature as the exogenous petro-hydrocarbons

and were not 3ffecred by the large concentrations of the foreign compounds.

Stegeman and Teal (L973) found that the fat content of the animal was

proportional to the maximum ability to store the foreign hydrocarbon








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

virginica, 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; GalsCoff, 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 pm, (3) marine bacteria 1.0 2.5 pm, and (4) macroparticulate organic

matter 1 10 pm and larger. Data from Ward and Aiello (1973) on the mussel

ljytilus edutis, a lamellibranch like Crassostrea, imply that the gill is

a dual 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 pm in size without mucus (Haven and Morales-Alamo, 1970).

The first in vitro work on uptake by lam:ellibranchs showed that the

gill tissue is the most important site in the animal for free amino acid








and sugar uptake (Bamford and Gingles, 1974; Bamford and McCrea, 1975).

By excising gill issue from. Ccrastoderma edule, the common cockle, anc'

measuring the uptake of 1C labeled amino acids, these workers demon-

strated that the uptake mechanism is saturable, has a diffusion co.jponent,

and that there is inhibition by other amino acids. Their work with the

Japanese oyster, Cra.sostrea 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 P6quignat

(1973) on the uptake. of. amino acids and glucose by Mytilus edulis. In

these whole animal experiments, labeled amino acids, removed from sea

water concentrated in tissues of the mantle, foot, and gills, i.e., those

soft tissues exposed to organic in the water as it passed through the

shell. It is obvious that the gill is vitally important in the feeding

process both for larga mac:rci.olecular aggregates and detritus in filter-

feeding and for direct assimilation of dissolved material.

The metabolic importance of la.nrllibranch 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 02; this rate of filtration may

then be expressed as 15 lites H 20.1 ml 02. If 1 ml of 0, will oxidize

0.8 mg of organic matter, and if the basal metabolism represents approxi-

mately one-third of the total oxygen consumption, the amount of organic

matter that must be removed from the sea water is

0.8
Sx 3 = 0.16 mg/liter.
15

Nicol suggests that the particulate diet of oysters, detritus and phyto-

plankton, can provide 0.14 2.8 mg of organic matter/liter. Since the

results of in vitro and in zivc uptake experiments with lamellibranch








molluscs (Bam&ford and Gingles, 1974; Bamford and McCrea, 1975; PAquignat,

1973; Stephens, 1963), show that dissolved material,present in concentrations

up to 10 mg/ml, can be removed from sea water, dissolved organic matter shot

be considered as a possible source of metabolic energy.


Research Objectives


The purpose of this research was to study the uptake and incorporation

of dissolved free fatty acids by a marine filter-feeding mollusc, the

American oyster, Crassostrea 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 what is the free iatty

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 of 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 does the uptake of dissolved material (i.e., smaller than

0.45 pm in diameter) compare with the uptake of particulate

material 50 pm 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 AND METHODS


Materials


Animals


Oysters of the genus and species Crassostrea virginica were

collected from an estuary on the west coast of Florida north of Cedar

Key known as Shell Mound. An area of collection was chosen which was

accessible without a boat and at mean low tide was covered with two-

uhree inches of water. The experimental plot was sheltered from heavy

boat. traffic and was exposed to a minimum of pollution due to the unpopu-

oted ar, arrnv.und it. Animals cf 7 10 cm shell length (2.5 3.5 grams

soft tissue weight) were selected at low tide and only 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

equipped 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 were used from July through

May because chose collected during the early summer were small and

gravid, frequently releasing eggs into the holding tanks or during the

uptake experirnnts.








Chemicals'

14 14 1
[1-- C]palmitic acid, [3- C]stearic acid, [16- C]palmitic acid,

and [7,8-"H]oleic acid were purchased from Schwarz-Mann. All non-

radioactive fatty acids were reagent grade and were recrystalized before

use. Standard samples of phospholipid and neutral lipids for thin layer

chromatography (TLC) and fatty acid methyl ester standards for gas

liquid chromatography (GLC) were purchased from Applied Science, Supelco

or Sigma Chemical.

Petroleum ether for extraction, column chromatography and TLC was

purchased from Eastman Chemical or City Chemical of New York, and glass

distilled two times over potassium permanganate. It was separated into

30 600C and 60 780C boiling fractions and was stored in dark bottles.

Aquasol was purchased from New England Nuclear and spectral grade

toluene PPO-POPOP was made with reagents purchased from Sigma Chemical

Chloroform and methanol for extractions were purchased from Eastman

Chemical as analytical reagent grade solvents and were not redistilled

prior to use. Anhydrous diethyl ether was purchased from Mallinckrodt

and was not redistilled prior to use.

All other organic and inciganic chemicals were analytical or

reagent grade.


Silanization of glassware


All glassware for uptake experiments, extraction, transporting and

storage of lipid material in aqueous or organic solvents was treated

with an aqueous silanizing reagent, "Siliclad," purchased from Clay

Adams, Inc.








Column packings for GLC


EGSS-X and Apiezon-L column packing 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 pm thickness on 20 x 20 cm

glass were obtained from E. Merck. Silica Gel G with no binder was

obtaJnpd from Applied Science and was spread on glass plates.


Column chro~matography


Specially prepared 400 nesh 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.


Se.- water collection and filtration


Sea water used in the uptake experiments was collected from the

Cedar Key estuary along with 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 Whatman #1 paper under vacuum

to remove large particles and then filtered through a Whatman GF/A glass

fiber filter of 0.45 pm porosity. The filtered sea water was stored in

glass at 40C until used as an uptake medium. Sea water to be extracted

for background free fatty acid levels was sampled as soon as the filtration

steps we'c' completed.








Methods


Uptake E:perinents


Closed shell experiments


The oysters to be used were removed from the holding tank and cleaned

of all epiphytic and epizoic material with an oyster knife and a heavy

bristle brush. They were then rinsed clean of all sand and left until

the shells were dry.

The labeled fatty acid was added to a small glass petri dish and the

carrier solvent, usually benzene, was removed with a stream of N2 gas.

A Teflon stirring bar was placed in the petri dish and the dish was

placed in a six-liter glass vessel. Four liters of bacteriologically

filtered sea water containing 200 mg/liter of streptomycin sulfate was

added with stirring. The sea water was sampled by removing 1 ml aliquots

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-

ether (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 involved 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 wl cf the cthanol-ether or 200 p! of the chloroform extract was

added to Aquasol and counted.








Oyster (remove from shell)


Count __Wash in 300 ml of sea water saturated with
3 ml aliquot palmitate


Weigh (to nearest 0.1 gram)


20 ml 20--------------


Homogenize 15 seconds in Waring blendor


Decant into 25 x 150 mm centrifuge tubes with
2 rinses (volume % 40 ml)


Ia


Add 70%
HC104 to make 0.6 M
4


Allow protein to precipitate (5 minutes at room
Temperature)


Spin in TEC centrifuge 2000 rpm :: 10 minutes


//7// Muscle mat (discard)


Supernatant discardd)


Pellet

Add 40 ml Bloor Reagent: FtOH-ether, 3:1


Allow protein to percipitate, centrifuge (IEC)
2000 rpm x 10 minutes

Count -- supernatant4 "'S pellet (discard)
1 ml aliquot


Figure 3. Extraction -ith Adapted Bloor Method.











Count <
1 ml aliquot


Oysters (3-4, remove from shell)

1.
Wash in 100 ml of sea water saturated with
fatty acid


Weigh oysters on pan balance to
(should be near 10.0 grams)

dI


nearest 0.1 g


Adjust weight with H20 to equal 10.0 grams


10 ml CHC1 3__
20 ml M -eOH -
20 ml MeOH

"


Homogenize
slow speed


10 ml CHC13 ---- -


Homogenize
high speed

I
Vacuum filt


Retentate
(discard)

V


90 seconds in Waring blendor at






30 seconds in Waring blender at



er through Whatman I# paper


Filtrate (allow to settle into two layers,
record volumes)


Count
200 p1 aliquot


4,


-- Aqueous methanol


- CHC13


Count 200 1il aliquot. Retain for incorporation
studies.


Extraction with Adapted Bligh and Dyer Method.


Figure 4.






Open shell experiments


For those uptake experiments 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 velve

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 c.vity

were used as a test of viability and successful removal of the upo'ur shell.

The animals were rinsed in sea water and then placed in the ve';sel con-

taining 4 liters of filtered sea water. At zero time :-nc labeled fatty

acid and any competing fatty acid dissolved in ethanol were added to

4 liters of sea water below the surface of tl.t vortex created by a stirring

bar, ensuring that the ethanol and the f0cty; a, id HcLt uijperCrcd rapidlyy

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 acscrbed on celite was studied using

Johns-Mansville celite sieved to approximately 50 pim size particles. The

fatty acid in appropriate concentration in ether solution was added to








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 N2 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 were taken and the procedure outlined in

Figure 3 or 4 followed. The sea water was sampled both unfiltered and

after 0.45 pm filtration to determine the concentration of free fatty

acids, and therefore, the degree of dissociation of the fatt3 acid from

the celite particles.


Temperature dependent uptake experiments


The temperature of the sea water solution vas maintained using a

copper-coil cooled/heated water reservoir around the 6 itear glass vessel.

The cooling or heating water in the coil was circulated from a Forma

Scientific water bath. The temperature of the sea water was thermo-

statically maintained with + 1 C of the desired temperature. The studies

of uptake were the same as described previously.


Turnover experiment


The animals were prepared as for the uptake experiments but the

shells were not removed. The oysters were placed in a glass vessel with

4 liters of filtered sea water. Seven mg of sodium [ H]acetate (5 mCi)

was added to the sea water. After 18 hours, the animals were removed,

washed in sea water, and placed in a glass vessel with 4 liters of

non-radioactive sea water. 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.








Aliquots of the extract were separated on TLC plates and counted and

quantitated.


Resolution cf Lipids


Thin layer chromatography


The neutral lipid classes were resolved by thin layer chromatography

techniques and identified by comparison with standard compounds. The

250 pm silica gel plates were divided into 2 cm channels and activated by

heating for 30 minutes at 1200C. Lipid extracts (100 or 200 pl) 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 PE/EE/HOAc (petroleum ether (30 600C)/dicthyl ether/acetic acid)

solvent (90/10/1) for approximately 1 hour. The solvent was removed

with a -treae of air and the material on the plate ;:A. visualized with

cither iodine vapor or by charring with sulfuric acid (Mangold,

1960). This TLC solvent system completely resolved the neutral lipid

classes of Crassostrea and the sea water extracts into stercls, tri-

glycerides, alkyl diglycerides, wax enters and sterol esters.

The phospholipid classes were resolved on silica gel plates

activated for 30 minutes at 800C, and developed in a chloroform/methanol/

waLer solvent (65/25/4) for approximately 80 100 minutes (Wagner,

1961). The plates were channeled and the extracts spotted in the same

manner as the neutral lipDds.

A better separation of the phospholipids could be achieved when the

neutral lipids were first removed from the extract by column chromato-

graphy over a 1 x 10 cm Hi-Flosil column. The extract, in chloroform,

was applied at the top and all the neutral lipids eluted with 2 column








volumes of CHC1.. The phospholipids were 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 in the polar lipid TLC system described previously.

For complete resolution of phospholipids, a two-dimensional method

was used in which the plate was developed in chloroform/methanol/water/

28% aqueous anmonia (130/70/8/0.5) in one direction and chloroform/

acetone/methanol/acetic acid/water (50/20/10/10/5) in the 900 direction

(Parsons and Patton, 1967).

Table 3 lists the visualization reagents which were employed in

the identification of the neutral anc pihcs poliipid compounds.

These reagents, together with a saponification step for esterified

compounds (Stahl, 1969), permitted the identification of the lipids found

in the oysters.


Quantitation of lipid material


The lipids following separation by thin layer chromatography were

quantitated using the method of Amenta (1964). The lipid on the TLC

plate was visualized with I, vapor and scraped into glass tubes; 1 cr 2

ml of a 8.5 pM solution of potassium dichromate in concentrated sulfuric

acid were added. The 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 the 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 at 350 nm,

comparing against a water blank. The difference in absorbance between a









Table 3. Visualization Reagents for TLC.


Reagents


12 Vapor


Chromic Acid-
Sulfuric Acid

Rhodamine B


Ni nhydrin-Butanol




Chromic Acid-
Glacial Acetic Acid
(1:1)

Amnmoniu:n Molybdate




Hydroxyl tLine-
Fcr.ic Chloride


----


---- -----


Function


General Screen


General Screen


General Screen


Ami no-Phospholi pid
and Glycolipids
Containing Glucosainine

Cholesterol
Cholesterol Esters


Phospholipids




Esterij iej Carbo-ylac
Acids


Reference


Bettschart and
Fluck, 195G

Bertetti,
1954

Kaufmann and
Budwig, 1951

Fahmy
et al., 3961


M; chalec,
3956


Hanes and
Isherwood,
1949

Whittaker and
Wijesundera,
1952


I-- ------------------- -








standard tube and a sample tube was compared to curves for cholesterol,

tripalmitin, cholesterol stearate, dimyristyl phosphatidyl choline, and

palmitic acid.


Scintillation counting


The aqueous samples of sea water, wash, and filtered sea water from

the uptake experiments were counted in Aquasol (1 ml aqueous sample added

to 10 al scintillant). The chloroform and methanol layers cF the -.xtiacted

material were counted in Aquasol at 200 ul/10 ml to reduce quenching of

the organic solvents. All samples were counted in a sub-ambient Packard

Tricarb at O0C and compared to suitable standards. The doubic label

experiments were counted in a refrigerated Nuclear Chicago counter in the

double label mode.

The radioactive lipids, once separated on TLC plates, were either

counted directly in a Packard TLC radiosca-ner or the lipids were scraped

off the plates directly into 5 ml of Toluene FOPOP ana counted in a

refLigerated 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 background and

counting efficiency by coincidence counting with [ C]k.olueie,
14 3
[4C]benzoate, and [ 3H]water standards purchased from Packard Instruments

and diluted as required.


Fatti Acid Methylation--GC Separation


Preparation of methyl erters


The fatty acids were methylated according to the method of Stoffel








et al. (1959). To the fatty acid samples separated by thin layer

chromatography were added 4 ml of 0.24 N IIC. in methanol and 0.5 ml of

dry benzene. The solution was refluxed at 80 1000C for 2 hours in a

ground glass apparatus fitted with a CaC12 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 600C) and this extract was dried over Na2SO4 and

NaHCO3. The petroleum ether was added to a sublimation apparatus (a side

arm test tube fitted with a cold finger) and evaporated. Then the fatty

acid methyl esters were sublimed in 200 pm vacuum and at 60 + 20C. The

methyl esters were 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 HC1 in methanol. The solution was allowed to react at room

temperature for 30 minutes and then, after the addition of 0.4 ml 1120,

was extracted 3 times with 2 ml of petroleum ether (30 600C). The

extracts were pooled, washed with H 0, evaporated to dryness, and

redissolved in hexane before injection into the GC.


Saponification and methylation


The direct saponification and methylation of fatty acids in the

lipid extracts were performed using a modification of the procedure of

Christopherson and Glass (1971). The lipid extracts were added to Teflon-

capped tubes and the solvent evaporated to dryness with N2 gas. Five ml

of 2 M potassium hydroxide in methanol solution were added and heated at








40 500C for 30 minutes. After the addition of 6 ml of water, the

solution was extracted 2 times with 5 ml of petroleum ether (30 600C).

The ether solutions were pooled, evaporated to dryness, taken up in

200 500 ;l of hexane, and stored in small 1 ml vials with Teflon-lined

screw caps. Aliquots (5 10 "il) 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 N2 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 isothermally at 180 C. This column resolved the 16-C and 18-C

series of fatty acid esters, but 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 2750C at 1.50C/minute 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 1900C with a 60 ml/minute

flow rate. At 1740C 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 all acids reported in 10-C -

22-C range can be resol'-d 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 ;-.ater

was extracted as described in the section on methods and fractionated by

thin layer chromatography. The results of the neutral anrl phorpholipid

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 21 and March 31 extracts and their presence in the chloro-

form extract of the October 31 sample can be attributed to the ise of

petroleum ether (30 600C) for their extraction. Jeffrey has shown 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 was justified.

14
Preliminary experiments with C labeled fatty acid revealed that better

than 90 percent extraction of the label could be effected with 1 extraction

step with petroleum ether (30 -- 60C) and 3 subsequent washes of the

extract with 2 N HC].

By comparison of the lipid extracts with known standards, those lipid

classes which are separated by TLC can Le idrt.jfied and quantitated by

the methods previously described. The results appear in Table 4. For the

June 21 extract the majority of the lipid appea's to be in the frac fartty














S / ) Cholesterol











) O -Phosphatidyl Ethanolamine







i Pliusphatidyl Cioline







9 Lyso-phosphatidyl
Choline



3 5
1 2 3 4 5


Figure 5. Separation of Polar Lipids in Sea Water Extracts.
Sea water was extracted with petroleum ether (June 21) or
chloroform (October 31) and 200 pI aliqucos r'u on the polar
lipid TLC system. 1, BFSW (bacterially fi:lter-e sea water)
from Oct. 31; 2, NBFSW (non-bacterially filtered sea water )
from Jur.c 21; 3, BFSW June 21; 4, cholesterol standard;
5, phospaholipid standard with standards listed on the right
margin. Dotted line at the top: solvent front.














(~7~


CO
0'










K-)
O
0
0
0



1


O






0





0


Figure 6. Separation of Neutral Lipids in Sea Water Extracts.
Sea water ,as, extracted with petroleum ether and 200 pi
aliquots run on the neutral lipid TLC system. 1, NBFSW
June 21; 2, BFSW from June 21; ?, BFSW from March 31;
4, standard n,:utral lipid mixture with components listed
in tlea right margin. Solid line at the topwasthe solvent
front.


0







/ \

Li


- Sterol Ester




- Fatty Acid Ester






- Triglyceride



- Free Fatty Acid

- Diglyceride
- Sterol
- Polar Lipids


0







0



0
0


3
3








Table 4. Concentrations of Extractable Specific Lipids in the Sea Water
Collected on June 21, 1974 (Extract A) and March 31, 1975
(Extract B).



Concentrations in Sea Water
Rfa Lipid Class in pg/l

Extract A Extract B

0.04 Monoglyceride Trace Trace

0.06 Sterol 14 32

0.12 Diglyceride Trace Trace

0.21 Free Fatty Acid 77 56

0.36 Triglyceride 31 47

0.65 Alkyl Diglyceride 3( 38

0.88 Sterol Esters 13 62

0.94 Hydrocarbons 104 53


Total 275 28



Relative migration of lipid class on a neutral lipid chronato-
graphic system relative to the solvent front migration.








acid and hydrocarbon fractions; together they comprise greater than

50 percent of the total lipid. The concentration of the free fatty acid,

77 pg/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 chrormatograph 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 thesedata, 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 methylation 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 water 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.



























-.i
0
O4





44
o
10
a)








a)
41
0)

.H


U)

"0
0 r

*4-




4-1




0
U)


OCU



0
4 U





Sa
0 C
r







U
00 1


0W

4a

C) r
00C





-a





4r- a-
0 4-
'u)



4-a


a) -0

0 -'I
*H U-











4a1
i)l (


" 0


" 80
U 3
01 *





S)


C *



0d C)

c- i c
H DJ


a
4J




a o





0
W I








c O
C O








AM m
oU cU -.






0





04d
-4





4 0
c) c'O
0) 4


0> 0


MU
oH 0W







Q) > 0
,-X Wr-i





00
4-1 4- 1-1





-r
I u



) 0 -
IJ 1 r--
N' 0 Nr
H"l)H




37

















^c"'






-4






00C










CO
r-1











CI *Hr



__---____-_--_-__________ --










1 C

























osuodsa-4 'jO4
r--I
O ----




































N
.IJ~
























ss0dsi I ljJ~Z'L








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.

Retention Time
Carbon Number Percent Composition
in Minutes

C-12 2.2 27.6

C-14 3.5 8.7

C-16 6.1 35.5

C-18 10.8 22.1

C-18:1 12.1 2.8

C-18:2 16.0 1.0

C-20:0 20.2 2.3








Uprake of Palmitic Acid


The ability of oysters to remove palmitic acid from natural sea

water solution was investigated with [-1- 4Cpalmitate at a concentration

-7
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 amount of isotope and carrier used. In each set

of expcrinmnts sea water from the same sample was used throughout to

minimize ary differences in salinity which might have affected the

uptake. Stephens has 'hown 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

organic which are not present in artificial mixtures, but which may

affect uptake processes, would be present. With artificial salts, in

the: quanLiti~: needd to make a 28 parts per tlousaind salt solution,

organic contaminants will be present in large concentrations compared to
-7
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 satisfactory 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 wash was employed, but we found this severely

dehydrated the animals and could possibly cause the removal of nore than

just adsorbed material. A wash procedure in sea water saturated with

the experimental fatty acid was found to exchange effectively any

simply adsorbed material (see Figiure 8). The loss of label could then

be monitored by sampling the wash solution at 30 to 60 minutes. In all

























































Figure 8.


1


n



a

a
u
a
,'
E, J'
0'


ij
ii
B


20 40 60 80 100


Time in Minutes


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 water was sampled in 1 ml
aliquots and counted in 10 ml of Aquasol. Animals were
labeled with 10 LCi 14C palmirate at a concentration of
2.8 x 10-7 M.


i n,_~-~-~, -y~i~PL~-~ rrrlZ1i~8sa








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-
-7
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 shown 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



















t*
0 01
S4J *3
O 0 Dn r-..
4- cd o
(0 4- -q 4-4
cc a) 0. 0


4 r 4 .-J C .

0 > 0H
C 0o OC )
04 0 01
-0 04-
Cfl 4-J*- J

C ,HCi M H


00
4 M4-J C

< Cd-W

-H 0 ir


4-J U) C7'
< S -H 00
0.Cd> CM
*H (0 r Ci)

S(C*i Cd




*U ( 4J C H
4d m 44 1
>, 4J *-,
C 4-1 rd -
o4 *M C:
*0 *H a
0d C ) 4J -1

4 0 0 0
ci d 0) 'Cd
cl 0 0 ca
1 -,,. r-1 C
C 0C) 0
0 0 Ct l ) "

4-' E4 Cd

-H 0 H 0C

400 .
to C JU J

4 J C 0
d C Cd )
4 J J rl 0 '

0< 60


P 4-J 4-W Cl
.C !> 0 0A
E OAw 0 P. c









< I
c

!
I----~ ---


I




I
-------: L--- ---


9 I
,I
I


4----


/1/


C,-)


Tma/uKaiO w OT
?-ul~i~ r 0~


U _________


|








(see Figure 10). In this control experiment less than 10 percent of the

labe] was removed from the water.

-7
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 adsorption

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 known 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/ether extraction step. A chloroform-

methanol extraction (Bligh and Dyer, 1959) as described in the methods

section was 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 the 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


















2.0 -


1.5






1.0


0.5 -


30 60 90 120


Time


in Minutes


Figure 10.


14
Removal of 1C 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 10-7 M palmitate with 10 uCi C
isotope and the shells of the same number of animals as
.normally used in the uptake experiments was plotted against
time. The shells were washed according to the methods
used for whole live animals.


c




















0
.11!
4--
0
C) zi 41



"0 r1 410
-J CI U C)



C-) 0
41 -H (1)
IU



W 4-H

*-7 3 E -H


S0

co 0 C




0 00
-, C) 4-) C


0 0 04 O

41 41 41 U 4



0C *-1
-tJ 00
C 0 4J.0


SH H U o)
a a o i-i



C)U
4-' o t M C


0 -r-1


0 0 r- ro

4- i > Ii6
4 4-I 00 *




- 10 co 0
0Zl 01



C) 'l .0 U) o

,--t C) ,I (
co if ^ -




47










I o

















.o
i







a E



A 0
O-(












e
CN








/
e










/ !




N, N


[I/u. ? x QOT








dispersing 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
-7
in ethanol, at a concentration of 2.8 x 10- M palmitatee) 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 counts in the lipid extracts were caused by the periodicity of

valve opening and closing in the animal's normal feeding cycle, but the

data show that during the first 2 hours almost all the label is removed.

The presence of CO2 in the sea water, shown 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 breakdown 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

animals exposed to air during the tidal cycle. When experimental




49






!(A) 50





Add Label Add Label 40
2-













20

a o
.e












e .
















The radioactivity in aliquo of the sea water (B), the
lipid extract ,and CO2 i an aliquot of sea water (C)
were plotted against time. The concentration of palmitate
in the sea water was 2.8 x 10-7 M after the first addition
at time zero and 2.8 x 10-7 after the second addition. A
Slabel as added in ethanol. 4C2 was


counted aftir traping int haine hydrode and adding to
ATuasol. The ivpid was exlutr the chloreform/me thanol

were plotted and against time. The concentratissueon f palchmitate
total of 20 Ci of 14C ise.tope as used; 10 Ci at each

counted after trapping in hyamine hydroxide and adding to








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 synchronization 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 animal 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

-7
temperature dependence of palmitate uptake at a 2.8 x 10 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 Upt3ke Experiments


The major assimilatory pathway utilized by the oyster is filter

feeding via the ciliary apparatus of the gills. While uptake of free



































(A)


-t -e *

<*
*
. l'ml







*
o
*
*
*
*




I.
2 0 6 810
20 40 60) 8"0 100


- 50







-40








H.
310
-30 u







-20









\-


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 were1 placed in 4 liters of sea water with a paImitate
concentration of 2.8 y 10-7 M and containing 10 pCi total
14C isotope. The lipids were extracted by the chloroform/
methanol method and the dpm/mg wet weight of the oysters
plotted.


. r...........
W























S----350C

.. 25 C


I!

/


. ................. ...... 30C


120


180


240


Time in Minutes


Figure 14. Temperature Dependent Uptake of Palmitate.

The radioactivity in the lipid extract of the animals was
plotted for three different temperatures. The concentration
of palmitate was 2.8 x 10-7 M with 10 PCi total 14C isotope
in all experiments. Each point was the average of two experi-
ments at each temperature. The results for 200C were
negative to 300 minutes. The lipids were extracted with
chloroform/methanol.


tI~I~M~LI;L~P -~-~'---~---iR-*-~-- = --- ----r~-- --


psn LL*nr r








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 apparatus.

In the autoradiographic work by Pequignat (1972), the labeled amino

acids which were taken up from the sea water by Myti.lus 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 pm

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 4 liters of sea water and the extent nf uptake

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 which the

particles were suspended were extracted with petroleum ether and the

absorbance at 525 nm (the maximum for oil red 0) determined. The results,

shown 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 [14C]palmitate was investigated. Ten pCi

of 1C labeled fatty acid was adsorbed onto 50 mg of 50 pm celite

particles with successive ethyl ether evaporations as described in the








Table 6. Localization of Oil Red 0 Celite Particles
Water by Experimental Animals.


Removed from Sea


Localization Time of First Absorbance (525 nma)
Localization of
Celite Particles Appearance of Pet Ether Extract
(Minutes) of Sea Water

Sea Water 0 0.299

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 spectrophotometer.








methods section. The uptake of this labeled celite was investigated

with whole animals. Figure 15 shows 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

45 minutes when compared with the uptake of similar concentrations of

-7
freely soluble palmitate at 2.8 x 10 M. This delay has been seen in

every celite particle uptake experiment run with oyster 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 jMtiZus edulis, the

label appearing in the gut much later than that which appears in the soft

tissues.

The concentration of free acids in the sea water was determiined by

the dpm/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.

-7
The uptake of celite-adsorbed palmitate at 2.8 x 10-7 M was also

investigated using the open shell animals (Figure 16). There is a dif-

ference between their accumulation of lbel and that in the whole

animal experiment. The organism can remove the label very efficiently

and at a linear rate up to 90 minutes. If this celite uptake is compared

to the uptake of 2.8 x 10-7 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 two different sea water

concentrations as dpm/ml of sea water//ap/n/mg of animal tissue in the

















2 -40




4-j
-30 -
Q)


S(A)


S- 120









..- *... .............. ...............................(c)



60 120 180 240


Time in Minutes


Figure 15. The Uptake of Celite-adsorbed Palmitate.

The total radioactivity in a 1 ml aliquot of the sea water
(B), a 1 ml aliquot of 0.45 pm filtered sea water (C), and
200 ul of the chloroform extract of the animals (A) was
plotted against time. The concentration of palmitate used
to prepare the 50 m-- celite was 2.8 x 10-7 M. Three animals
were extracted at each point.










10 -50






8 -40













4 20
\ /
6 .\ -30













2 / (B)L10




A






Time in Minutes


Figure 16. The Uptake of Celite-adsorbed Palmitate, Open Shell Animals.

The radioactivity in 1 ml aliquots of the sea water (B),
1 ml 0.45 uim filtered aliquots of the sea water (C), and
aliquots of the chloroform extract (A) were plotted against
time. The conceitralion of palmitate used to prepare the
celite was 2.8 x 10-7 M. The animals were added after the
removal of the upper valve. Three animals were sampled at
each point.
























4-1
ctn
^ 4-4
oH con
H! Cd C
M) 0* 4-1
CC






c) *H *U
44 4J.






C d*H *H
)(U -0 -
4-J 4 -i aU

OT-Cr C3
l *rl 4-1
o3 C) *U *>-


r) e o 6



S0 Cd T
'O > 4-1


10 00



U 0 r-1 c*'r-l


4*H- 0

I4-J ) c(
4-i 3C d



i ct *-io
CO 40 4J









o 00 a) r
4- 4-J 44



Cd OT D *

0o W u
0 U O)T c

i 0 (4 44l (U j
rq r- c'Li
o 4i) c oT -:




















0 0

0 0


-


















-i.


0 e

c4
S*
0
0
0
o
0 *


0
00
o


0 0


0 0

a
0




0 0


0 0



0


0
0
0


0 *
0

o o
0

o "
0 3

0
0
0


0

0
0 0











0o *

0oo
00*







00






. c . . .****** .+.....+. . . .*. ..






0 0 0 0 0 0
0 4












0 0 0 0 0 0
In (*j *


jo3'2j UOT u2iJ3ouoD X OT








chloroform extract. From Figure 17, the celite uptake for open shel.l

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 would 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-rate. 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


The concentration dependent uptake process was investigated with
14
open-shell animals and C labelled palmitic, stearic, and oleic acids.

The incorporation of [ C]palmitate 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 were computer plotted by least squares. See

Figures 19 and 20. The slopes of the plots of the initial rate of uptake

is plotted versus concentration. Figures 21 and 22, the saturation plots


























CJ-

SU



S4






*0





00




-H




r-









UC)
0 0
aO












- ,41f
.)ZCQ























*lIl,

















t



*
0 0


0 0 -

0
0 .
0 *
0
0
o a



0 *


0 0
C *
0 0 c

0
0 *


o0


0


S. 0
f z




0 0



0 .
o o



o o 0
o -n


0


o*
0
0
0 *


0 0 *

oo
0 0
o0 0
o o 4r t




CC*
o e


C-




o
0

0 ,
0
0

0 o



0
)0 a


- .4 .............4+ +....... S C .+........ 4........ .... s o






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Figure 21. The Concentration Derii 1ent Rate of Uptake of Palmitate.

The initial rate of uptake determined from the slopes of
Figure 19wereplotted against the concentration of palmitate
in the experiments. The animals had the upper shell removed

prior to addition to The sea water.




68







0i











0.41




















3-0 2.0 3.0 4.0 5.0

10 7 Concentratiooi (I)


Figure 22. The Concentration Dependent Rate of Uptake of Stearate.
The initial, rates of uptake determined from the slopes of
Figure 20wereplotted against the concentration of stearate
in 5 experiments. The animals had the upper shell removed
prior to placement in the sea water.








for palmitate and stearate, show similar saturations at low concentra-

tions, then a sudden burst in the uptake rate appears at 3.0 or

7
6.0 x 10 M. This is probably due to self-aggregation of the fatty

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 micellar concentration of

palmitate to be about 5 x 106 M. In the experiments with natural sea
-7
water reported here the micellar concentration is about 7.0 x 10 M.

The difference in the two figures emphasizes the importance of considering

the 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
6.0x 1-7
6.0 x 10 M, i.e., below the aggregation concentrations, has a y

.intercept, Km of 5.0 x 10 M1, and a maximal velocity of 0.78 dpm/mg/min.

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
-7
is 0.59 x 10 and the maximal rate of uptake is 0.63 dpm/Tag/min. 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 10-7 M







*1 *










/ 6 e .




1 '
2 ; /








2


/

0 4 8 12 16





S
1/





















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 Kr for the uptake process from the slope (V = dpm/mg
wet weight/min) (S = 10-7 M omitting the point at
8 x 1C-7 M).





















0~
C
r
r
r
r
E
O

L
d
C
Y

()

0.*
r
d
C

,

r
r


Figure 24. Lineweaver-Burk Transformation 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 Figure 23.

The rate for S = 4.2 x 10-7 M was omitted.


~ru~rrnu- --- ----Y-----DC--~1P~s ~-~rr~lW


V


I







-7
for palmitate and 0.60 x 10 for stearate. At naturally occurring

concentrations the oysters are able to remove both palmitate and stearate

from the water because palmitate is below the half-saturating concentration

and stearate is about equal to the half-saturating concentration. Other

data on the fatty acid distribution indicate that the levels of palmitate

may represent a greater percentage of the total free fatty acid and

stearate a lower percentage for other areas and methods of determination

(Jeffrey, 1970). Our evidence then indicated that the animals had a
-7
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-
-7
trations from 1.25 15.0 x 10 M. The initial rates of the uptake are

shown in the computer plot of least squares velocities in Figure 25.

The velocities are only linear for the first 30 to 45 minutes and show a

saturation at longer times. When the initial rates of uptake are plotted,

a linear relationship is found with no saturation even at a 1.5 x 10-6 M

concentration. (See Figure 26.) The ambient concentration of oleate

-9
in 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. From 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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Lipids of Crassostrea and the
Incorporation of Labeled Fatty Acids


The neutral lipids of Crassostrea virginica have been characterized

by column chromatography and thin layer chromatography (Watanabe and Acl-Jan,

1972). We found 5 major classes of neutral lipids as can be seen from a

TLC of the lipid extracts from a [14 Cpalnitate 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 palmitate uptake

experiment in Figure 28. The 2 major compounds are those with relatJve

mobilities of 0.3 and 0.63, phosphatidyl choline and phosphatidyl

ethanolamine, respectively.

The genus Crassostrea, unlike the genus Ostrea, 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 estcrified

lipid or catabolized for energy. Also, there is no problem 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 at., 1S69))

By determining the incorporation into specific lipids, the actual uptake

and incorporation rates can be measured and quantitated.

The radiochromatographic scans of the neutral and polar lipid
-7
separated by TLC following a 2.8 x 10 M palmitate uptake experiment are

shown in Figures 29 and 30. Superimposed un the scans are the traces of


















































2 3 4 5 6 7 8 9


Figure 27. The Thin Layer Chromatographic Separation of Oyster Neutral
Lipids.
The lipid extracts from a 2.8 x 10-7 M palmitate incorporation
experiments were run on che 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 alkyl
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 palmnit-te, and cholesterol oleate. The dotted
line at the top of the plate was the solvent front.








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1 2 3 4 5 6 7 8 9

Figure 28. The Thin Layer Chromtagraphic Separation of Oyster Polar
Lipids.
The lipid extracts from a 2.'8 x 10-7 palmitate incorporation
experiment were run on the polar lipid ILC system parallel
with stancird mixtures. The lipids were visualized with
iodine. (I 7): 200 4l 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-phosphatidvl
choline, pliospatidyl choline, phosphatidyl ethanolamine, and
cholesterol. The dotted line at the top was the solvent front.


















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the lipids visualized by iodine vapor. In the neutral plate a large
14
amount of 1C activity was seen at the origin, representing incorporation

into the pnospholipid material. 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 mere

polar solvent system in which the sterols and the phcspholipids were more

completely resolved. Very little incorporation was seen in the alkyl

diglycerides 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 aPd 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 ethanolaminc (see Figure 31).

The fatty acid distribution in 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 TLC, and the individual compounds

which showed activity in the radiochrnomatographic scans 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 thaE over 90 percent of the activity was located in the

phosphatidyl choline with the remainder found in phosphatidyl ethanolamine.




85












........................ .... ........................................ ..................................................... r' ......


It










0 \
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00




Figure 31. The Two-dimensional TLC Separation of Oyster Phospholipids.
The 120 minute extract of a 2.8 x 10-7 M palmitate uptake
experiment ,7as run in the two dimensional solvent system
desciibeJ in the mechods. The separation achieved in the
first solvent system was 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 il of
the chloroforim extract. The so!l-ent fronts were shown by
the dotted line.
























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PAGE 1

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

PAGE 2

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 execution 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.

PAGE 3

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

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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 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

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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

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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

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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 Incorporation 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 Incorporation 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

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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 extractable 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.

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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 concentration 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

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INTRODUCTION Metabolic Signif jcance of Disgolved Orf^anlc Ma 1 1 er The salt waters of the world contain relatively constant concentrations 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.

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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 leakage 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 substances (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 environmental forces involved in the production and processing of organic .atter by the benthxc animal communities are as complex and as important as the Liochemical 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.

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Light v.yL Living Organic ) Matter " 4ApsimiJaLion i. Excretion I Filter \ > Dying and \ Feeding \ V,xcr<^.tion \ Assimilation of Dissolved Organic Matter \ Exc Overp Excretion and \ verproduction \ \ ^ Decomposition Rain

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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) .

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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 metabolically 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

PAGE 15

ths nutrition of lnvcrttlo„ 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 important 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 concentrations, were removed from sea water solutions by several marine species (.,ee Table 1). Eased on results with radioactive tracers, the

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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

PAGE 17

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 metabolic 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.

PAGE 18

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.

PAGE 19

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 concentration in the medium. The ability to remove dissolved amino acids and carbohydrates at isoionlc sea water concentrations, however, is real and their net movement

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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 Fa tty 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

PAGE 21

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^

PAGE 22

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

PAGE 23

14 material. This would seem to indicate that, once removed, the material mixes with the lipid pools of the organism. The Oyster as a Possib le Experimental Subject for Lipid Upt ake 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) nanoand 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

PAGE 24

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 demonstrated 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 filterfeeding 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 approximately onethird of the total ox-ygen consumption, the amount of organic matter that must be removed from the sea water is 8 YJx 3 = 0.15 mg/liter. Nicol suggests that the particulate diet of oysters, detritus and phytoplankLon, can provide 0.14 2.8 mg of organic matter/liter. Since the results of in vitro and in vivo uptake experiments with lamellibranch

PAGE 25

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?

PAGE 26

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 twothree inches of water. The experimental plot was sheltered froir. heavy boat traffic and was exposed to a minimum of pollution due to the unpopulated 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 filtration 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

PAGE 27

18 Chemicals l^^l__-._.... -_.. ro lA^. _._ 1^ [l-"^ C]palraitic acid, [3Cjstearic acid, [16-" Cjpalinitic acid, and [7,8-"H]oleic acid were purchased from Schwarz-Maun. All nonradioactive 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^ilaniza tio n 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.

PAGE 28

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. Co lumn chroma tography Specially prepared 400 mesh silicic acid for lipid column chromatography was purchased from Bio-Rad. Hi-Flosil, a silicic acid derivative for rapid separation of lipid classes, was purchased from Applied Science. Sea water collecti on 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.

PAGE 29

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 ethanolethtr (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.

PAGE 30

21 Count 1 ml aliquot 20 ml II 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.

PAGE 31

22 Count
PAGE 32

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 containing 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 concentration 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 experimen t 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

PAGE 33

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 experi ments 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 thermostatically maintained with + 1 C of th^ desired temperature. The studies of uptake were the same as described previously. Ta rnover 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.

PAGE 34

25 Aliquots cf the extract were separated on TLC plates and counted and quantitated. R esolution 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, triglycerides, 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 chronatography 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

PAGE 35

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:! ra tion 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

PAGE 36

Tablo 3. Visualization Reagents for TLC. Reagents Function Reference I^ Vapor Chromic AcidSulfur ic Acid General Screen General Screen Bettschart and Fluck, 1956 Bertetti, 195A Rhodamine B Ninhydrin-Butanol Chromic AcidGlacial 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 /uniueFcrric Chloride Esterifieu C.irboxyiic Acids Whit taker and Wijesuiidera, 1952

PAGE 37

28 standard tube and a sample tube was compared to curves for cholesterol, tripalmitin, cholesteryl stcarate, dimyristyl phosphatidyl choline, and palmitic acid. Scintill ation coun tiuR 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 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 Separatio n P repar ation of me t hyl er t ers The fatty acids were methylated according to the method of Stoffei

PAGE 38

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 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. S aponif ica t ion 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 Tefloncapped 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

PAGE 39

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 Chr om atography 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).

PAGE 40

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 chloroform 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

PAGE 41

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.

PAGE 42

^:) \ a r u o o Q o o O ^-v-^ / O O 8 O O o r.\ O 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.

PAGE 43

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;

PAGE 44

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 predominant 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.

PAGE 46

37 c a c •H d o c 0) 4-1 Pi 3Suods3vi Jo^oa^eQ

PAGE 47

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

PAGE 48

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 thewash solution at 30 to 60 minutes. In all

PAGE 49

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.

PAGE 50

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 solubilizers 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 concentration 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

PAGE 51

(U

PAGE 52

43 M C_) , (0 (U c g •H H Tin/mu'O X 01

PAGE 53

(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 addition 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

PAGE 54

45 e B a •a X I O 2.0 0.5 1.0 30 60 Time in Minutes — r90 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. *

PAGE 56

47 n \ \ / \ ^ / ^ O IT) o r o ON o o 1-° ,-»„Jj w G U 3 C •H :£; c •H 0) B •H H -[UI/UJCO X Q-^^ c

PAGE 57

dispersins the acid. The problems of variability of initial label concentration 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

PAGE 58

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.

PAGE 59

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 concentration 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

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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.

PAGE 61

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 experiments at each temperature. The results for 20°C were negative to 300 minutes. The lipids were extracted with chiorof orm/methanol.

PAGE 62

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 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

PAGE 63

54 Table 6. Localization of Oil Red 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. 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.

PAGE 64

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 nonparticulate 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 indicating 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 difference 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

PAGE 65

56 26 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.

PAGE 66

s? 10 50 £ I o 82. ^ 40 •30 (A) \ ^: 4 / / •(c) 20 40 — r60 80 to 0) 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.

PAGE 68

59 < P3 o o o o o o o ® o c. O o e . CO o o o " o O o o e « • • • •^^ in O C ir, O CO C •H H O O o c o o o o acrto'Pj; uoT3Fa.3U3Duo3 x qt

PAGE 69

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

PAGE 71

62 ffi PQ 7j4ueouoo x OT

PAGE 73

6^ c c w © ,T1 ^ — ' o * o 9 O o o o o c o o « o <6 o e ® -K o o • o o f o o CO o 8 ' + • c c l/^ L-l CM C CO (U 4-1 c •r-l i-i o OJ en o O CO o auSjaAv 53ft Si'i/uidp SF a>ju:idn

PAGE 74

CO C

PAGE 75

w «>

PAGE 76

2.0-1 1.6. 1.2, 0.8. O.A -i 67 e-' ..*• 2.0 4.0 6.0 8.0 10 X Concentration (H) 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.

PAGE 77

68 •H g M e 1.00) 4-1 p. ]=> o •H O o 1-1 a; > 0.80.6•H C M 0.40.2-5 I / 1.0 2.0 3.0 4.0 5.0 10' X ConcentrafiOii (M) 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..

PAGE 78

69 for palmitate and stearate, show similar saturations at low concentrations, 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 solution. 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

PAGE 79

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 palmitate 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),

PAGE 80

71 31 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.

PAGE 81

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 concentrations from 1.25 15.0 x 10 NThe 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.

PAGE 83

74 c o w u * — y n < ?J» x OP fk ^\ |T3-idn

PAGE 84

in CNJ u 60 4-) o 4^ d i) c -o c o c o o c o 4-1 c o y o u 4-1 Cfl to en U-l o O O a s: 4J E O ^^ «-i -a (1) c •H 6 i-i CJ 4J o •X} 0) to 4.1 o. CO ,1: j-i 4-) c e -H •rl H 4J Q) C o 4-) ca c c o a 0) w M 3 4-1 1) y) ^ C 4.) •H rt XI cd ^ T) en 4-1 n) 4-1 B O -rj ex n) o c-.i l^

PAGE 85

Ih O >:; c o u C o c o o ujm/Sui/mdp ut. ai^E^dn _;o A^f-^oxaA T^'PT"!

PAGE 86

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 intracellular 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

PAGE 87

-"> c? CD c:d 8 c^ r\ O o 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 cholesteryl 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.

PAGE 88

79 I CO O U o "A O o o CD O O) o o (3 u. o o ~1 o 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 dimyristyl 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,

PAGE 90

81 j._o c o •H A-l CO M 60 •H > to o

PAGE 92

83
PAGE 93

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 phosphatidyl 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 experiments indicated that over 90 percent of the activity was located in the phosphatidyl choline with the remainder found in phosphatidyl ethanolamine.

PAGE 94

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 ethanolamine in the extract. The origin was spotted with 200 yi of the chlorororm extract. The solvent fronts were shovTi by the dotted line.

PAGE 96

87 vO rjuBAjos (0 0) *J 3 (3 •H CD •H H C O •H 4J C (U •U 0) as asuodss^ joTosaaQ

PAGE 98

89 00 m 0) 4-1 3 C •H s C (U e •H M O •H 4-1 iJ O Pi 3suods9)j jo^oarjBQ

PAGE 100

9J m o [ o 00 \ / / / / n — r CSl \ o CM O SO V \ w to C) 4-1 3 c •H ;^ c pTd-fx JO Srl/radp ui uoT^-Baodaoouj

PAGE 101

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 incorporated/r.iin, a saturation plot is obtained (see Figure 36). The LineweaverBurk 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 incorporation 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.

PAGE 103

OA o o o o o o o o o *® O (I o o o o o X o o o > o o o o
PAGE 104

95 CO e
PAGE 105

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) .

PAGE 106

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 assimilation 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.

PAGE 107

98 •H o p. CO o 1^ o H 00 "b o •H 4J CO o a. o u H 5 .0 A.O 3 .'} 2.0 1.0 0.0 A (C) (A) e (B) -•3 O O o ^' ° A o o » . o o o o » o 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 phospholipid 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 concentrations 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.

PAGE 108

99 •H E 60 e 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.

PAGE 109

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).

PAGE 110

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 0.91 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.

PAGE 111

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 demonstrated. 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 aggregation of these acids then promotes the uptake of the included palmitate as was seen in the data for the uptake of large concentrations of palmialone (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 palmitate 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 frothe inhibition experiments the results must be interpreted very c-refully. If a small inhibition is seen it

PAGE 112

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 0.88 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.

PAGE 113

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 3.6 2.5 X lO"'' M 2.8 X lO"'' M 16.8 5.0 X lO"'^ M 13.5 5.0 y. 10~ M 2.8 X lO"^ M 15.9 Rates measured in least squares slope of initial velocity plots.

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lOf may ba entirely due to dilution effects. At large fatty acid concentrations, 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 investigate 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 preliminary 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

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106 600 500 ' •H & 60 e (X 400S \ 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 artificial 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.

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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.

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109 PQ U / / t / / / CO o o O -o CM o o 05 c •H c •H Hi e pTdT-f Srl/mdp

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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

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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 concentrations (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' filterfeeding 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 autoradiographic 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

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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

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113 all the lipid classes, but the major incorporation occurs into the phospholipids, 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 incorporation into the triglycerides varied from one experiment, and even from one group of animals, to the next. The large turnover rate seen for the triglycerides 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 thsmall 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

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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 normallyremoves 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;

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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 pathways 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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BIBLIOGRAPHY Amenta, J.S. J. Lipid Res. 5:270-272 (1964), Anderson, J.W. , and G.C. Stephens. Mar, Biol. 4:243-249 (1969). Applied Science Laboratories, Inc. GasChrom Newsletter. 14(5) :3 (1973) ^^'"^°(i;74if"' ^"' ^' '''"^^^"^^"^Bioohem. Physiol. 49A:637-6A6 Bamford, D.R. , and R. McCrea. Comp. Biochem. Physiol. 50:811-817 (1975). '''''9b);i20~124'(1962)?"" ^"' ^''^ "^^^^^^^l^^^^P ^ea Res. Baylor, E.R., and W.H. Sutcliffe. Limiol. & Ocean. 8(4):369-371 (1963). Bertetti, J. Ann. Chim. (Rome) 44:495 (1954). Bettschart, A., and H. Fluck. Pharm. AotaJ^alv. 31:260 (1956). Bligh, E.G., and W.J. Dyer. Can. J. Bioohem. Physiol. 37:911-917 (1959). Bloor, W.R. J. Bio. Chem. 77:53 (1928). ^^'^''''(1972)' ^"^' ^^^P^^""^' ^""^ ^•^' "ealey. Biol. Bull. 142:219-235 Christopherson, S.W., and R.L. Glass. J. Dairy Sai. 55:1289-1290 (1971). ''"'''S;/*;.';"'' ^ll ^•^•,^^P:;it^ky, and J.O. Rell. Fish. Bull. 84. t%sh. Lu.l. oj the Fxsn and Vlildlife Ser-o. Vol. 54 (1953) . Duursma. E.K. N etherlands J . of Sea Res. 1:1-147 (1961). Fahmy R.A. , A. Niederwieser, G. Pataki, and M. Brenner. lielv C Ur Acta. 44:2022 (1961). f 'uiti . neoo. LLim. Farkas, T. , J.C. Neven.cl, and A. A. Benson. L^pid^. 8:728-731 (1973). Ferguson, J.C. Biol. Bull. 138:14-25 (1970); 141:122-219 (1971). Fossato, V.U., and E. Sivieru. Mar. Biol. 25:1-6 (1974). ^°''' (i9S3)i^'^' ^^P^"'"'''^^"' ^^^d -J-SKxttredge. J. mr. Res. 12:233-243 116

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117 BIBLIOGRAPHY—Continued Galstoff, P.D. Fish. Bull. 64. U.S. Govt. Printing Ofc, Wash. D.C. (1964). Garrett, W.D. Deep Sea Res. 14:221-227 (1967). Gilles, R. 5 and E. Schof f eniels. Comp. Biochem. Physiol. 31:927-939 (1969). Hammen, C.S. Am. Zool. 309-318 (1969). Hanes, C.S., and F.A. Isherwood. Nature. 164:1107 (1949). Haven, D.S., and R. Mo rales -Alamo. Biol. Bull. 139:248-264 (1970). Hobble, J.E., C.C. Crawford, and K.L. Webb. Science. 159:1463-1464 (1968) Hoshi, M. , M. Williams, and Y. Kishimoto. J. Lipid Res. 14:599-601 (1973) Jeffrey, L.M. , B.F. Pasby, B. Stevenson, and D.W. Hood. In Pro c. Int. Mtg. (Milan) (1962). Jeffrey, L.M. JAOCS. 43:211-214 (1966). Jeffrey, L.M. Lipids of I'.ar. Waters. Gee. Pub. #1:1-625 (19 70). Johannes, R.E., and K.L. Webb. Science. 150:76-77 (1965). Johannes, R.E., S.J. Cov/ard, and K.L, Webb. Comp. Biochem. Physiol. 29:283-288 (1969). Johnr.on, R.G. J. Mar. Res. 32(2) :313-330 (1974). Kaufmann, H.P., and J. Budwig. Fette u. Seifen^ Antriohmittel. 53^390 (1951). Korringa, P. Quart. Rev. Biol. 27 (4) :266-365 (1952). Kritchevsky, D., and S. Malhotra. J. Chromatog. 52:498-499 (1970). Krogh, A. Biol. Rev. 6:412-442 (1931). Lee, R.F., K. Sauerheber, and A. A. Benson. Science. 177:344-346 (1972). Little, C, and B.J. Gupta. J. Exp. Biol. 51:/59-773 (1969). Little, C. , and B.J. Gupta. IJature. 218:873-874 (1968). Lucas, C.E. Synrp. Soo. Exp. Biol. 3:336 (194Q) ; 15:190-206 (1961). Lucas, C.E. Biol. P^,v. 22:270-295 (1946).

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118 BIBLIOGRAFHY—Continued Mangold, H.K. "Aliphatic Lipids" in Tkin Layer Ckromatogra'phy . Springer-Verlag, N.Y. pp 363-420 (1960). Michalec, C. Bioohem. et Siophys. Acta. 19:187 (1956). Nicol, J. A. The Biology of Marine Animals. Wiley Intersclence, N.Y. (1970). Okaichi, M. Kagcwa Daigaku riogakubu Gakujutsa Hokoku. 18(2) : 181-185 (1967). Parsons, J.G., and S. Patten. J. Lipid Bes. 8:696 (1967). Pequignat, E. Mar. Biol. 12:28-41 (1972). Pequignat, E. Mar. Biol. 19:227-244 (1973). Putter, A. Vie Emahrung der Wassertiene und der Stoffhaushalt der Gewasser. Fisher Jena, Germany (1903). Reish, D.J., and G.C. Stephens. Mar. Biol. 3:352-335 (1969). Riley, G. Linmol. & Ocean. 8:372-381 (1963). Ryther, J.D., W.H. Dunstan, K.R. Tenore, and J.E. Huguenin. Eio-Soience. 22(3):14A-152 (1972). Shick, J.M. Biol. Bull. 144:172-.179 (J973); 148:]T7-140 (1975). Slowey, J.F., L.M. Jeffrey, and D.W. Hood. Geochim. Cosmochim. Acta. 26:607-616 (1962). Slowey, J.F. , L.M. Jeffrey, and D.W. Hood. In Preprints Int. Ocean. Congr. pp 934-937 (1959). Southward, A.J., and E.G. Southward. Sarsia. 45:69-96 (1970); 48:61-70 (1971); -0:29-46 (1972). Stahl, E. Thin Layer Chronatcgraphy. Springer-Verlag, N.Y, (1969). Stauffer, T.B., and W.G. Macintyre. Chesapeake Sci. 11(4) : 216-220 (1970) Stegeman, J.J,, and J.M. Teal. Mar. Biol. 22:37-44 (1973). Stephens, G.C. Biol. Bull. 123(648-659) (1962); 126:150-162 (1964). Stephens, G.C. Cctnp. Biochen. Physiol. 30:191-202 (1963). Stephens, G.C, andR.A. Schinske. Biol. Bull. 115:341-342 (1958), Stephens, G.C., and P. A. Schinske. Lirmol. 6 Ocean. 6:175-181 (1961). Stephens, G.C., and R.A. Virkan. Biol. Bull. 131:172-185 (1966).

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119 BIBLIOGRAPHY— Continued Stoffel, W., F. Chu, and E.H. Ahrens. Anal. Chen. 31 (2) :307-308 (1959) Sutclifi'e, V:.H. , E.R. Baylor, and D.W. Menzel. Deep Sea Ees. 10:233-2A3 (1963). Taylor, A.G. Comp. Biochem. Physiol. 29:243-250 (1969). Tenore, K.R. , J.C. Goldman, and J. P. Clarner. J. Exp. Map. Biol. Eool. 12:157-165 (1973). Testerman, J.K. Biol. Bull. 142:160-177 (1972). Thomas, J. P. Mar. Biol. 11:311-323 (1971). Wagner, F.S., Jr. Contrib. in Mai>. Soi. 1A:115-153 (1969). Ward, M.E., and E. Aiello. Physiol. Zool. 46:157-167 (1973). Watanabe, T. , and R.G. Ackman. J. Fish Res. Bd. of Ccniada. Tech. BuJl. No. 334 (1972). Whittaker, V.P., and S. Wijesundera. Biochem. J. 51:348 (1952). Williams, P.M. J. Fish Res. Bd. of Canada. 22:1107-1122 (1965). Williams, P.K. Nature. 189:219-220 (1961).

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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

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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

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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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n1/> \/Uy\/v -TA ' .^^ \ 'jt ,,.^. ^^"^ ^ ^ 1B7 ^5 ^0 0.1.