Demonstration of the Deposition and Modification of Dietary Fatty
Acids in Pinniped Blubber Using Radiolabelled Precursors
Suzanne M. Budge
Margaret H. Cooper*
Sara J. Iverson
Department of Biology, Dalhousie University, Halifax, Nova
Scotia B3H 4J1, Canada
Radioisotopes are commonly used to study the in vivo metab-
olism and deposition of dietary fatty acids in adipose tissue.
The application of this approach to pinnipeds is problematic
because of their large mass and blubber fat content. We have
developed a method where labelled lipids can be fed to seals
at financially feasible levels, with the radioactivity in individual
fatty acids isolated from blubber detected with standard lab-
oratory equipment. A combination of techniques including ar-
gentation thin layer chromatography, high performance liquid
chromatography with ultraviolet detection, and independent
liquid scintillation counting were employed. Juvenile gray seals
(Halichoerus grypus) werefed either0.5mCi3H-labelledtriolein
(18:1n-9,) or palmitic acid (16:0,
n p 2
ples were taken 12 h later, and the radioactivity in individual
fatty acids was determined. Radioactivity was detected in only
18:1 from the animals fed3H-labelled triolein, indicatingdirect
deposition without modification. Both animals fed3H-labelled
palmitic acid showed clear peaks of radioactivity in 16:0; how-
ever, there was also significant activity (23%–29%) found in
the desaturation product 16:1. Our results demonstrate that
this method is sufficiently sensitive to track the deposition of
labelled dietary lipids as well as modification products of in-
gested fatty acids and will be important in the application of
fatty acid signatures to study predator diets.
). Blubber sam-
n p 2
*Corresponding author; e-mail: firstname.lastname@example.org.
Physiological and Biochemical Zoology 77(4):682–687. 2004. ? 2004 by The
University of Chicago. All rights reserved. 1522-2152/2004/7704-3088$15.00
There is considerable interest in the use of blubber fatty acid
composition to study diet in marine mammals, especially pin-
nipeds (seals and sea lions; Iverson 1993; Ka ¨kela ¨ et al. 1993;
Iverson et al. 1997a, 1997b, 2004; Smith et al. 1997; Brown et
al. 1999; Kirsch et al. 2000; Walton et al. 2000). Recent work
has demonstrated that fatty acid signatures can provide quan-
titative estimates of predator diets provided that the differential
metabolism of individual fatty acids within the predator is ac-
counted for (Iverson et al. 2004). Weighting factors are used
to provide a composite picture of the influence of differential
deposition, modification, utilization, and de novo synthesis of
individual fatty acids on the overall fatty acid composition of
blubber. The relative importance of these processes varies with
the fatty acid composition and fat content of the diet, poten-
tially altering the values of these weighting factors. The direct
investigation of the modificationanddepositionofspecificfatty
acids provides insightintotheunderlyingbiochemicalprocesses
and can be used to understand the effects of variation in diet
on these factors.
Desaturation and chain elongation of dietary fatty acids such
as 16:0 as well as chain shortening of very long chain mono-
unsaturated fatty acids have previously been suggested to occur
in pinnipeds (Ackman et al. 1971; Ackman and Hooper 1974);
however, this was based on purely circumstantial evidence.
Studying the metabolism and subsequent deposition of fatty
acids in vivo requires the use of tracers. Radioisotopes have
been used successfully for such studies in small animals such
as rats and fish (Owen et al. 1975; Thomassen et al. 1985; Hjelte
et al. 1990; Linares and Henderson 1991; Green andYavin1993;
Rabinowitz and Myerson 1994; Nilsson et al. 1996). Pinnipeds,
however, present a significant problem in tracking ingested la-
belled fatty acids. Pinnipeds are large in body size, and blubber
constitutes a high percentage of body mass (approximately
10%–45%; Worthy and Lavigne 1987; Ryg et al. 1990; Mar-
kussen and Ryg 1992; Iverson et al. 1995; Arnould et al. 1996;
Aarseth et al. 1999; Kirsch et al. 2000); this results in a very
large dilution factor for the labelled fatty acid. To ensure that
the levels of radioactivity in the adipose samples are above
detection limits, mCi amounts of labelmustbeemployedrather
than the mCi doses typically used with small animals. Although
mCi doses have been used in studies involving humans, these
studies were not concerned with quantifying the radioactivity
in individual fatty acids but rather just the total radioactivity
in an adipose sample (Ma ˚rin et al. 1990). When the purpose
is to study the fate of specific dietary fatty acids, a propor-
tionately larger dose of radioactivity is required. The cost of
such labelled compounds canbe quitehigh.Therefore,studying
in vivo lipid metabolism in large animals such as pinnipeds
requires the development of methods of analysis that are ef-
fective when relatively small doses (!1 mCi) of labelled dietary
lipids are used.
We have employed the fatty acid tracer method to study the
deposition and potential modification of specific dietary fatty
acids in the blubber of juvenile gray seals (Halichoerus grypus).
Our objectives were to develop a method of analysis that was
sufficiently sensitive to detect very small amounts of radioac-
tivity in individual fatty acidsinsmallblubbersamplesobtained
by biopsy sampling and to use this method to determine the
fates of two fatty acids common in diets and blubber stores of
pinnipeds. This excluded the use of convenient methods nor-
mally applied in fatty acid tracer studies such as gas chroma-
tography (GC) or high performance liquid chromatography
(HPLC) with direct radioactivity detection, simply because the
detection limits of those instruments are much too high to
allow the use of an affordable amount of tracer. We instead
employed HPLC with UV detection and independent scintil-
lation counting. Because HPLC separation is based on polarity,
fatty acid methyl esters (FAME) with differing chain lengths
and degrees of unsaturation will coelute. Thus, a preliminary
separation of FAME into groups with the same number of
double bonds via argentation thin layer chromatography
(AgNO3-TLC) was performed.
Material and Methods
Four free-ranging juvenile gray seals were captured on Sable
Island, Nova Scotia, placed in a fenced-in, covered enclosure
on the beach, and fasted for approximately 12 h. Each seal was
fed a diet of approximately 1 kg of ground fish spiked with
either 0.5 mCi tritium-labelledtriolein(
1n-9, Mandel Scientific, Ontario) or palmitic acid (
[9,10-3H]16:0, DuPont NEN, Perkin Elmer Life Sciences Can-
ada, Ontario) by gastric intubation. We used only two animals
for each study since our main purpose was to develop the
analytical methods for tracer feeding and recovery and to val-
idate the identification of modification products. The body
composition of the two seals fed
timated from measurements of total body water using deute-
rium dilution methods (Bowen and Iverson 1998) and predic-
tive equations developed for gray seals (Reilly and Fedak1990).
After 12 h, blubber biopsies were taken from both the right
and left flank of each animal according to Kirsch et al. (2000).
This time period was chosen in light of the rapid rate of passage
of digesta in pinnipeds (Helm 1984; KrockenbergerandBryden
1994) and prior evidence of chylomicron clearance from the
,[9,10-3H(N)]18:n p 2
,n p 2
3H-labelled triolein was es-
blood of juvenile gray seals by 12 h postfeeding (Cooper et al.
2003). Blubber obtained from each animal totaled approxi-
mately 0.5 g. The animals were then released. Experimentswere
approved by the Dalhousie University Committee on Labora-
tory Animals under protocol 98-016.
The decision to feed 0.5 mCi of labelled fatty acid was based
on the known detection limit of our scintillation counter and
The detection limit of the liquid scintillation counter was ap-
proximately 40 dpm, corresponding to
ing a fat content of 22% (an average value for the age class of
grayseals[ ]usedinthisstudy;M.H.Cooper,unpublishedn p 31
data), a 40-kg juvenile gray seal should contain about 8.8 kg of
blubber, which would produce an approximately 9,000-fold di-
lution of the ingested radioactivity on a per gram blubber basis.
Under the best case scenario (i.e., all labelled fatty acid is de-
posited in the blubber), an excess of
to be fed to exceed the detection limit when analyzing 0.5 g of
fat. Since one should never plan for the best case scenario, we
Lipids were extracted from the blubber according to a mod-
ified Folch et al. (1957) procedure described in detailbyIverson
et al. (2001). Briefly, samples were extracted with 2 : 1 chlo-
roform : methanol and dried over anhydrous sodium sulphate.
FAME were formed by reaction of approximately 100 mg of
lipid with 1.5 mL of boron trifluoride in methanol (8% v/v)
and 1.5 mL of hexane. The mixture was heated at 100?C for 1
h under nitrogen, and FAME wereextractedintohexane.FAME
TLC (Rezanka 1996). Preparative silica gel plates (250 mm coat-
ing thickness, cm, Sigma Aldrich) were dipped in 20%5#20
AgNO3in acetonitrile and heated at 110?C for 1 h to remove
all water. FAME samples (approximately 10 mg) were evapo-
rated to near dryness and spotted on the plate in a single broad
band. The band was focused into a narrow line (approximately
1 cm from the base of the plate) by repeatedly developing in
9 : 1 hexane : diethyl ether until the bottom of the band mi-
grated to the top edge of the original band (approximatelyfour
repetitions). Plates were dried under a stream of nitrogen be-
tween developments. Plates were then fully developed in 9 : 1
hexane : diethyl ether until the solvent front reached the top
of the plate. Plates were again dried, sprayed with dichloro-
fluoroscein, and visualized under UV light. Seven bands were
apparent, each representing a group of FAME having the same
number of double bonds (from 0 to six). The bands were then
scraped from the plate and extracted with 1 : 1 hexane : diethyl
ether. GC was used to confirm the purity of each extract.
diameter, 5 mm particle size, Sigma Aldrich) and a 200-mL sample
loop. A flow rate of 0.8 mL min?1of 95% aqueous methanol was
used for all separations except that of the saturated FAME, for
mCi would have
S. M. Budge, M. H. Cooper, and S. J. Iverson
Figure 1. Distribution of radioactivity measured in fatty acids isolated
from blubber of each gray seal fed 0.5 mCi of
([9,10-3H(N)]18:1n-9). Plotted horizontal line represents background
1983). FAME were detected with UV absorbance at 205 nm.
Typically, fatty acids are analyzed as their phenacyl derivatives
because these stronglyUV-absorbingfunctionalgroupsincrease
the detection limits of the instrument (Christie 1982). Ouraim,
however, was to separate and recover fatty acids with as large
a sample throughput as possible, so detecting trace amounts
of lipid was not a concern. The use of FAME rather than their
phenacyl derivatives also eliminated a step in sample prepa-
ration, thus preventing the potential losses associated with ad-
ditional sample manipulations. Sample throughput was con-
strained by the column bore, which allowed the injection of
only 1 mg FAME peak?1. For a typical fraction containing 10
mg FAME, four to five injections would normally be required.
Eluents were manually collected in glass test tubes, and all
fractions containing the same individual FAME were pooled.
Each pooled fraction was then evaporated under nitrogen until
only the water portion remained. Approximately 2 mL of iso-
propyl alcohol was added, and then both water and alcohol
were evaporated. FAME were taken up in hexane and dried
over anhydrous sodiumsulphate.AfterGCanalysis,eachFAME
was mixed with scintillation cocktail (ScintiVerse I) and
counted in a Beckman Scintillation Counter (LS3801).
GC was used to determine FAME compositions before and
after HPLC using a Perkin Elmer Autosystem II Capillary GC
equipped with a flame ionisation detector and a flexible fused
silica column (30 mm inner diameter) coated withm#0.25
50% cyanopropyl polysiloxane (0.25-mm film thickness; J & W
DB-23, Folsom, Calif.). Helium (flow rate 1 mL min?1) was
used as the carrier gas, and the gas line was equipped with an
oxygen scrubber. The following temperature program was em-
ployed: 153?C for 2 min, hold at 174?C for 0.2 min after ramp-
ing at 2.3?C min?1, and hold at 220?C for 3 min after ramping
at 2.5?C min?1. Hydrogen and air had flow rates of 45 and 455
mL min?1, respectively. Both the injector and flame ionisation
detector were isothermal at 250?C and 270?C, respectively.Split
injection (100 : 1) with a sample size of 1 mL was employed.
FAME were identified by comparison of retention times with
known standards (Nu Check Prep, Elysian, Minn.).
Results and Discussion
AgNO3-TLC separation of FAME generated seven bands, with
each band predominantly containing FAME of the same degree
of unsaturation. With some samples, GCanalysisrevealedslight
one more or one less double bond than expected. Recoveries
from the TLC plate were on average
likely occurring when the bands were scraped from the plates
and transferred as a powder into the extraction vialscontaining
1 : 1 hexane : diethyl ether. Despite the presence of potentially
interfering FAME of different degrees of unsaturation in each
fraction, coelution on HPLC was rarely a problem. Because the
interfering peaks were small, in most cases it was possible to
collect them as a well-defined shoulder on the larger peaks.
Masses of FAME before and after HPLC were very similar with
recoveries near 100%, indicating negligible losses at this step.
Figure 1 illustrates the results from the seals fed3H-labelled
triolein (18:1n-9). The 18:1 isolated from blubber clearly con-
tained radioactive tritium, thus verifying that the analytical
method proposed here is sensitive enough to track the depo-
sition of labelled dietary lipids. Although the level of radio-
activity in 16:1 from the blubber of Hg3416 was slightly above
background, it is not clear whether this was analytical noise or
a real effect of chain shorteningof3H-labelled18:1beforedepo-
sition. Considering the very small amount of radioactivity
found in 16:1, if chain shortening of 18:1 to 16:1 does occur,
it likely does not constitute a major pathway in the metabolism
of dietary 18:1. Future studies with additional animals and the
methods now developed will be important to assess possible
between-individual variability in these processes. Figure 2 il-
lustrates the data from the seals fed
(16:0). Both animals exhibited clear peaks of radioactivity in
16:0; in this case, however, there was also significant radioac-
tivity found in 16:1. This is the first direct measure of desat-
uration of the dietary 16:0 to 16:1 in pinniped blubber and
confirms our expectation of the activity of the common D9
desaturase enzyme. It also demonstrates that this method is
sufficiently sensitive to detect deposition of modification prod-
ucts (16:1) as well as the original ingested fatty acid.
The mass and percent total body fat of the seals fed
labelled triolein are presented in Table 1. Both seals were well
within the expected range for percent total body fat but above
the average value for their age class of gray seals (22%; M. H.
Cooper, unpublished data). However, because body mass was
less than 40 kg, the dilution of the radioactive fatty acid was
only slightly greater than predicted. The two triolein-fed seals
had body fat masses of 11.0 and 9.9 kg (Table 1), corresponding
to 11,000- and 9,900-fold dilutions of the ingestedradioactivity.
, with losses 85%?10%
3H-labelled palmitic acid
Figure 2. Distribution of radioactivity measured in fatty acids isolated
from blubber of each gray seal fed 0.5 mCi of
acid ([9,10-3H]16:0). Plotted horizontal line represents background
Table 1: Mass and body fat measurements of individual study animals
SealLabelled LipidMass (kg)
Body Fat Fat (kg)
Thus, if all 0.5 mCi of ingested 18:1 was deposited in the
blubber, we would expect to see ≥
or 99,880 dpm g?1blubber. On average, only 1,373.0?95.3
dpm g?1blubber was found in 18:1. This is equivalent to !2%
of the possible maximum.
Because we are confident that approximately 85% of the
radioactivity present in the blubber samples is recovered using
this technique (calculated from the AgNO3-TLC and HPLC
recoveries), this low recovery of radioactivity relative to the
possible maximum (!2%) is likely not due to flaws in the
chemical method employed. Rather, it appears that the vast
majority of labelled fatty acids, ingested with a small meal after
12 h of fasting, are not deposited in the blubber in the first 12
h after ingestion. A major portion of this ingested fat may have
been used to fuel immediate metabolic needs, particularly if
the labelled lipids are more readily oxidized than natural lipids,
which would be in more complex associations with other food
components being digested. It is not known whether a larger
proportion of radioactivity would have been measured in the
blubber had we fed a larger meal, used nonfasted animals, or
allowed a different period of time to elapse between ingestion
and sampling. Ma ˚rin et al. (1990) found that, in humans, the
tissue was incorporated within the first 24 h, with much of that
being accounted for within the first 4 h. It therefore seems
unlikely that sampling later than 12 h postfeeding would have
provided any significant improvement in the recovery of ra-
dioactivity. Thus, under the current feeding conditions, ouruse
of a quantity of radioactivity 1,000 times in excess of the pre-
dicted minimum requirement appears necessary rather than
The two seals fed3H-labelled palmitic acid differed dramat-
ically in the amount of radioactivity recovered per gram of
blubber sampled, with Hg1220 showing a much stronger ra-
dioactive signal in its blubber than Hg1222 (Fig. 2; 4,792.8 vs.
738.5 dpm g?1blubber for 16:0). Unfortunately, body com-
position was not measured in these animals,sothetruedilution
of radioactivity into blubber mass is not known. However, on
the basis of their mass (Table 1) and the average percent total
body fat of 6-mo-old gray seals, Hg1220 was expected to have
a radioactivity concentration 1.3 times greater than that found
in Hg1222. In actuality, Hg1220 had a concentration 5.7 times
greater than Hg1222. Thus, the smaller estimated fat mass and
correspondingly smaller dilution of ingested radioactivity in
Hg1220 can explain only a small portion of the total difference
in radioactivity recovered. It is possible that there was some
loss of the labelled fatty acid during the process of gastric in-
tubation. Despite differences in the recoveries of absolute
amounts of radioactivity from Hg1220 and Hg1222, therelative
proportions of 16:1 produced from 16:0 were similar (29% and
23% of3H recovered, respectively). These values may be of use
in the understanding and refinement of weighting factors for
16:0 and 16:1 used in estimating diets based on fatty acid sig-
natures (Iverson et al. 2004).
Our use of independent scintillation counting was justified
on the basis of the levels of radioactivity recovered in the blub-
ber. With the pooling of fractions containing the same indi-
vidual fatty acids, the maximum level of radioactivity detected
in any one fatty acid was approximately 7,000 dpm g?1FAME
for 16:0 in Hg1220. Because the HPLC column capacity was 1
mg FAME peak?1, the maximum level of radioactivity that
could have been detected had this HPLC been equipped with
a scintillation counting device was ∼7 dpm. This is below the
detection limit of radioflow detectors, which typically have
background levels around 10 dpm, confirming the impracti-
cality of such equipment for these purposes. Therefore, this
method of analysis improves the level of detection by approx-
S. M. Budge, M. H. Cooper, and S. J. Iverson
imately three orders of magnitude. This will be particularly
important for future studies using other labelled fatty acidsthat
can be as much as five to six times more expensive than those
used in this study.
The AgNO3-TLC-HPLC procedure described here is labour
intensive as a result of the multiple HPLC injections of the
bands recovered from the TLC plate. An obvious solution is
computer-automated sample injection and fraction collection,
allowing analysis to continue without human intervention. A
second possibility is prescreening of samples for radioactivity
after AgNO3-TLC separation. It is clear from Figures 1 and 2
that the polyunsaturated fatty acid fractions were not radio-
active. Had a small portion of these fractions been prescreened,
this absence of radioactivity could have been establishedbefore
the HPLC analysis, saving much time and labour. Clearly,how-
ever, the combination of AgNO3-TLC-HPLC with independent
scintillation counting allows the appearance of radioactively
labelled fatty acids to be accurately tracked in the blubber of
juvenile gray seals. Specifically, this method makes the in vivo
study of the metabolism of individual fatty acids in large mam-
mals financially feasible by allowing a reasonably small amount
of label to be employed. In addition, we have provided the first
direct evidence in pinnipeds of the deposition without modi-
fication of a monounsaturated fatty acid (18:1n-9) as well as
the importance of the D9 desaturase enzyme to the metabolic
fate of a dietary saturated fatty acid (16:0). The results of this
study and those of future studies of other important dietary
application of fatty acid signatures to estimate diets.
We thank Don Bowen, Jim McMillan, Ken Meade, and Julian
West for their contributions in the field. We also thank Cheryl
Craft of the National Research Council of Canada Institute for
Marine Biosciences fortechnical assistancewiththeHPLCanal-
yses. This study was supported by a Natural Sciences and En-
gineering Research Council of Canada (NSERC) postdoctoral
fellowship to S.M.B., an NSERC predoctoral fellowship to
M.H.C., and NSERC operating and equipment grants to S.J.I.
The Department of Fisheries and Oceans, Canada, provided
additional support for fieldwork.
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