Indian Journal of Geo-Marine Sciences
Vol. 42(1), February 2013, pp. 120-124
Elevated levels of saturated and unsaturated fatty acids highlight the nutritional
value of Ulva covalengensis, a marine dietary alga found in south India.
Vidyashankar, S & Krupanidhi, S*
Department of Biosciences, Sri Sathya Sai Institute of Higher Learning,
Prasanthi Nilayam -515134, India.
*[Email: [email protected]]
Received 18 July 2011; revised 12 November 2011
Gas chromatography coupled to mass spectrometry (GC-MS) is applied to examine the relative levels of saturated and
unsaturated fatty acids in two species of coastally-derived edible algae Ulva covalengensis and Enteromorpha flexuosa and
compared their profiles with those of non-edible algae Chaetomorpha antennina and Grateloupia lithophyla. Present study
reveals elevated levels of fatty acids in Ulva covalengensis that included higher levels of arachadonic acid, the precursor of
prostaglandin, source of energy and component of cell membranes.
[Keywords: Marine Algae, Fatty Acids, Gas Chromatography coupled to mass spectrometry]
Introduction
Sea weeds are used extensively as food in many
coastal countries like Japan, Korea, Indonesia and
in some regions in India1. They are nutritionally
valuable as they have high dietary fibres, proteins and
metabolites such as polyunsaturated fatty Acids
(PUFA) in appreciable quantities2. Algal fatty acids
(Omega-3-Fatty acids and PUFA) are beneficial and
act as prophylactic supplements for type-2-diabetes,
atherosclerosis, coronary heart diseases, arrhythmias
and cancer 3. Individuals consuming excessive
fish-related products are exposed to xenobiotic
compounds that occur as natural contaminants in
marine ecosystem. There is an increased interest in
utilization of sea weeds as primary source of
PUFAs in some parts of coastal India4. Many studies
have demonstrated the edibility of select marine
macrophytes like Enteromorpha flexuosa and Ulva
covalengensis2,5,6, which have been shown to contain
high levels of PUFA2,4. However, these PUFA’s
found in these dietary marine algae have not been
well characterized. Present study is initiated to
examine the levels of saturated and unsaturated fatty
acids in Ulva covalengensis and Enteromorpha
flexuosa and compare it with those found in nonedible algae namely Chaetomorpha antennina, and
Grateloupia lithophyla. This study measure levels of
saturated fatty acids namely myristate (C14:0),
——————
*Author for correspondence
palmitate (C16:0) and stearate (C18:0) and
unsaturated fatty acids namely palmito-oleate
(C16:1), oleate (C18:1), linoleate (C18:2) and
arachidonate (C20:4), using gas chromatographycoupled to mass spectrometry.
Materials and Methods
All reagents for GC-MS were procured from Sigma
or Isotech (both St Louis, MA) unless specifically
mentioned. GC-MS analysis was carried out
using a 6890N Gas chromatograph-coupled to mass
spectrometer. The column used for GC was 15-m
DB-5 capillary column (inner diameter, 0.2 mm; film
thickness, 0.33 micron; J & W Scientific Folsom, CA)
containing 5% phenyl. Derivitization of samples was
performed using Butyldimethyl-silyl-triflouroacetamide
(MtBSTFA, Regis Chemicals, Morton Grove, IL).
The algal samples used in this study were collected
from rock surfaces and washed once with sea
water and later with running tap water to remove
all possible epiphytes, salts and small zoophytes.
The samples were shade dried and powdered using
a blender and stored in air tight plastic containers till
further use.
Fatty acids were enriched using organic solvent
phase extraction. Specifically, 250 mg of each sample
was de-pigmented with 5 mL of 100% acetone
and dried. This was followed by a second round
of extraction using 5 mL n-hexane (HPLC grade,
Ranbaxy, S.A.S Nagar, Punjab). Isotope-labeled
VIDYASHANKAR & KRUPANIDHI : ELEVATED LEVELS OF SATURATED AND UNSATURATED FATTY ACIDS
(D3-Deuterium) fatty acids (myristate, palmitate and
stearate (Sigma, St.Louis, MO) were used as internal
standards to account for extraction, derivatization,
and column injection variations in the study.
100 pmoles of each of these standards were
incorporated into the samples prior to extraction.
The samples were sonicated at 20 kHz and
extracted overnight in organic solvents indicated
above with constant stirring. The lysates were
centrifuged and the supernatants were recovered
and stored in dark to prevent photo-oxidation.
To enhance the recovery, the sediments were
subjected to an additional round of extraction
described above. The extracts were pooled,
evaporated and derivatized for GC-MS analysis.
Gas Chromatography analysis of Fatty Acids
Gas chromatography (GC) is the most commonly
used technique for identification of fatty acids7.
It exploits the differences in the partition coefficients
of volatilized analytes between a stationary liquid
phase and a mobile gas phase as they transverse
through the column. To facilitate the same, fatty
acids are derivatized to increase their stability
and volatility8. Prior to derivatization, the algal
extracts were re-dried under vacuum desiccation
for a minimum of 24 hours. This was followed
by multiple rounds of azeotrope formation with
dimethylformamide (DMF, 100 µL) for complete
removal of any residual water content. Derivitization
was
performed
using
Butyldimethyl-silyltriflouroacetamide (MtBSTFA) under nitrogen
atmosphere. The resultant t-butyl dimethylsilyl
(DMTBS) derivatives of fatty acids were analyzed
using a 6890N Agilent GC-mass spectrometer
(Agilent, Santa Clara, CA) as described previously.
Specifically, GC-MS analysis was carried out
in electron impact mode at high resolution.
Samples were injected using an on-column injector.
During the course of the run, temperature was
ramped from 40° to 300°C in a 16 minute period.
GC was interfaced with an Agilent 5973 mass
detector. The t-butyl dimethylsilyl (DMTBS)
derivatives of fatty acids were quantified by
either selected ion monitoring (SIM, for saturated
fatty acids) or full scan (for unsaturated fatty acids).
In all cases the molecular ion peak for labeled
myristate was used as a reference to account
for technical variations in the assay. Also for
ease of interpretation, levels of fatty acids in
121
Ulva, Grateloupia and Chaetomorpha were
expressed as fold change relative to those in
Enteromorpha.
Results and Discussion
Figures 1 A-C shows the GC-MS derived parent
ion spectra for palmitate (16:0, m/z 313), myristate
(14:0, m/z 285) and stearate (18:0, m/z 341).
Compared to Enteromorpha, the levels of
both saturated fatty acids were higher in
Ulva, Grateloupia and Chaetomorpha, and showed
a differential profile between the three algal
species (Fig. 1D and 2). Specifically, palmitate
and stearate levels were highest in Ulva followed
by Grateloupia and Chaetomorpha which had
lower levels of the metabolite (Fig. 1D, black and
grey bars respectively). Myristate levels, however,
were similar between the 3 algal types (Fig 1D).
Among the saturated fatty acids palmitate and
stearate levels were found to be differential
between the 3 algal species studied while no
difference was observed in levels of myristate
(Fig. 1-D, blue bars).
Among unsaturated fatty acids (Figure 2), we
assessed levels of oleic acid (18:1, m/z-339.3,
Fig. 2A), arachidonic acid (20:4, m/z-361.3,
Fig. 2B), linoleic acid (18:2, 337.3, Figure 2C)
and palmito-oleic acid (16:1, trace of molecular
ion not shown). Similar to our observation with
saturated fatty acids, the levels of all the unsaturated
fatty acids were higher Ulva, Grateloupia and
Chaetomorpha
compared
to
Enteromorpha.
Interestingly, levels of arachidonic acid and oleic acid
were highly elevated (25 & 10 fold respectively,
Fig. 2D grey and black colour), in Ulva compared
to other algal species. The higher levels of these
fatty acids make these algae a rich source of dietary
energy and justify their consumption in coastal
regions, Chennai. Furthermore, in addition to
serving as energy resource, arachidonic acid
(AA, [Cis-5, 8, 11, 14-20:4], Omega-6) is one
of the vital components of cellular metabolism.
These are precursors in biosynthesis of regulatory
molecules such as prostaglandins, thromboxanes
and other eicosanoids in cells9. Also, along with
other C: 20 PUFAs such as eicosapentanoic acid
(EPA) and a C: 22 PUFA docosahexanoic acid
(DHA), AA form the major components of
phospholipids in the cell membranes of nervous tissue
of brain9.
122
INDIAN J. MAR. SCI., VOL. 42, NO. 1 FEBRUARY 2013
Fig. 1—GC-MS analysis of saturated fatty acids in marine algae. A) GC-MS trace showing the molecular ion for Palmitic acid with m/z:
313. B), GC-MS trace showing the molecular ion for Myristic acid m/z: 285 C) GC-MS trace showing the molecular ion for stearic
Acid, m/z: 341 D) Graph showing relative fold change of stearate (grey bar), palmitate (black bar) and myristate (blue bar) in
Ulva, Grateleloupia and Chaetomorpha compared to levels of the corresponding saturated fatty acids in Entermorpha.
VIDYASHANKAR & KRUPANIDHI : ELEVATED LEVELS OF SATURATED AND UNSATURATED FATTY ACIDS
123
Fig. 2—GC-MS analysis of unsaturated fatty acids in marine algae. A) GC - MS trace showing the molecular ion for oleic acid with
m/z: 339.3. B) GC - MS trace showing the molecular ion for arachadonic acid, m/z: 361.3 C) GC - MS trace showing the molecular ion
for linoleic acid, m/z: 337.3 D) Graph showing relative fold change of linoleic acid (grey bar), arachadonic acid (black bar) oleic acid
(magenta bar) and palmito-oleic acid (blue bar) in Ulva, Grateleloupia and Chaetomorpha compared to levels of corresponding
unsaturated fatty acids in Entermorpha.
INDIAN J. MAR. SCI., VOL. 42, NO. 1 FEBRUARY 2013
124
Conclusion
In the present study the levels of saturated and
unsaturated fatty acids were assessed in four sea
weeds derived from the southern coast of India using
GC-MS. The findings indicated significantly elevated
levels of saturated and unsaturated fatty acids in Ulva
covalengensis, a prime dietary source in the region.
Elevated levels of arachidonic acid, a critical
intermediary metabolite in humans, infers this marine
macrophyte as a useful nutritional component.
Acknowledgements
Authours thank Dr. T. M. Rajendiran from
Michigan Center of Translational Pathology,
University of Michigan, Ann Arbor, MI and
Dr. Arun Sreekumar from Baylor College of
Medicine, Houston, Texas for help with GC-MS
analysis of fatty acids. Authors thank Prof. V.
Krishnamurthy and Dr. Mrs. S. Chandra,
Krishnamurthy Institute of Algology, Chennai for
helping in sample collection and identification
and Dr. P.Reddanna, School of Life Sciences,
University of Hyderabad, for his scientific input.
References
1
Dhargalkar, V.K. & Kavlekar,D, in: Sea weeds - a field manual,
(National Institute of Oceanography, Goa). 2004, pp.1-36.
2
3
4
5
6
7
8
9
Burtin, P., Nutritional value of sea weeds, J. Environ. Agric.
Food Chem, 2 (2003) 498-503.
Doughman SD, Krupanidhi, S & Sanjeevi.C. B., Omega-3
fatty acids for nutrition and medicine: considering
microalgae oil as a vegetarian source of EPA and DHA.
Current Diabetes Reviews, 3 (2007), 198-203.
Bhaskar, N, K.T., Miyashita, K, Park SB, Endo, Y &
Fujimoto, K, Occurence of conjugated polyenoic fatty acids
in sea weeds from the Indian ocean,. Naturforsch, 59 (2004)
310-314.
Morales, MA, V.M., Dominguez, SC, Acosta, BG & Gil, FP,
Chemical Composition and microbiological assays of marine
algae Enteromorpha Spp. as a potential food source,.
Journal of Food Composition and Analysis,18 (2005) 79-88.
Fleurence J, C.C., Mabeau S, Maurice M & Landrein A,
Comparison of different extractive procedures for proteins
from the edible seaweeds Ulva rigida and Ulva rotundat,.
Journal of Applied Phycology, 7 (1995) 577-582.
Aitken, A., Mass spectrometric Techniques, in: Principles and
Techniques of Biochemistry and Molecular Biology, edited by.
Wilson, K & Walker, J (Cambridge University Press) 2005,
pp. 405-447.
Weintraub, S.T., Mass Spectrometry of Lipids, in: Mass
Spectrometry of Biological Materials, edited by McEwen,
C.N. and Larsen, B.S. (MARCEL DEKKER, New York)
1990, pp. 257-286.
Khotimchenko, S.V.& Gusarova, I.S., Red Algae of Peter the
Great Bay as a Source of Arachidonic and Eicosapentaenoic
Acids, Russian Journal of Marine Biology, 30 (2004)
183-187.