Fatty Acid Profiles of Sauger and White Bass from the Ohio River as Indicators of Subpopulations, Sp

1
Fatty Acid Profiles of Sauger and White Bass from
the Ohio River as Indicators of Subpopulations,
Species and Season

• Le-Ellen Dayhuff
• Ph.D. Dissertation

2
Acknowledgements

• Corps of Engineers
• West Virginia Division of Natural Resources
• Tennessee Technological University Center for the
  Management, Utilization and Protection of Water
  Resources

3
Outline

• Introduction
• Factors Affecting Fatty Acids
• Analytical Chemometric Methods
• Ohio River Mainstem Study
• Conclusions

4
Why Study Lipids Fatty Acid Composition in Fish?

• Fish Health Nutrition
• Forensic Purposes
• Environmental Indicators
• Nutritional Implications
• Potential for Pollutant Bioaccumulation

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Example Fatty Acids (FAs)
Stearic Acid (180)
hydrophilic
hydrophobic
Docosahexaenoic Acid (226n-3)

• Nomenclature based on
• chain length (1st number)
• degree of unsaturation (2nd number)
• position of double bonds (3rd number)

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Degree of Saturation

• Saturated FAs (SFAs)
• Monounsaturated FAs (MUFAs)
• Polyunsaturated FAs (PUFAs)
• n-3 FAs
• n-6 FAs

First double bond occurs 3 or 6 carbon atoms from
terminal methyl group
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Environmental Effects on FAs

• Season
• Temperature
• Location
• Aquaculture
• Water Quality

8
Analytical Methods

• Sample Preparation
• Lipid Extraction
• Hydrolysis
• Derivatization (Esterification)
• Analyses
• Gas Chromatography (FID-GC)
• GC - Mass Spectrometry (GC-MS)
• Electron Impact (EI)
• Chemical Ionization (CI)

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Extraction of Lipids

• White muscle homogenized in
  chloroformmethanol
• Vacuum filtered
• Filtrate transferred NaCl added
• Refrigerated
• Na2SO4 added
• Filtered

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BassOMatic
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Hydrolysis of Triacylglyceride to Fatty Acids
Triacylglyceride Glycerol Fatty Acids
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Derivatization (Esterification)FA ? Fatty
Acid Methyl Ester (FAME)
FA FAME
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GC-FID
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GC Standards and QA/QC

• Multi-components FAME standard
• Analyzed with each set of samples
• Used to calculate correction factor K(t)
• Acceptable K(t) values 0.80 1.20
• Maintenance needed if K(t) outside acceptable
  range

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Total ion gas chromatogram of FAMEs identified in
a sauger fillet
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Fatty Acids Identified in Freshwater Fish
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GC-MS

• Designed to do 3 things
• Converts neutral molecules into beam of positive
  ions
• Separates ions on basis of mass-charge-ratio
  (m/z) ratio
• Measures relative abundance of each ion
• From this information we can
• Determine molecular weight formula
• Obtain clues about molecular structure

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EI MS

• Sample molecules bombarded with high-energy
  electrons (70eV)
• Collisions result in loss of e- from molecule to
  form positive ions
• MS detects only cations
• Too destructive for highly unsaturated FAMEs
  (HUFAs)

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CI MS

• Sample introduced with excess methane reagent gas
• Ionizing electrons from carrier gas ionize
  methane
• CH4 e ? CH4. 2e
• CH4. CH4 ? CH5 CH3.
• CH5 M ? MH CH4
• Produced MH ions for all FAMEs
• Less fragmentation than EI
• Common ions M1, M-1, M-31, M-33

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GC-MS Analyses
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Docosahexaenoic acid 226n-3 ME Mass Spectra
from Sauger
EI MS
CI MS
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EI Fragmentation

Burhenne, J. Parlar, H. A Contribution to the
Identification of Fatty Acids from Fishoils by
Mass- and FTIR-spectroscopy. Fresenius Environ.
Bull. 1993, 2, 119124.

McLafferty, F.W. Mass Spectrometric Analysis.
Molecular rearrangement. Anal.Chem. 1959, 31,
8287.
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CI MS of Vaccenic acid (181n-7) ME
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EI MS of Vaccenic acid (181n-7) ME
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Ohio River Mainstem StudyGoal of Grant

• Does fatty acid profiling constitute a suitable
  biomarker?
• Are there differences in the fatty acid profile
  that can be explained by other than population
  isolation or homogeneity?
• Can fatty acid profiling tell us about the
  fitness of fish?

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Ohio River Study Sites(Ohio River 981 miles from
Pittsburgh to Mississippi River)
Sampling performed within 1000 feet below dam
that compromised upstream boundary of pool
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Species Sampled

• Sauger (Stizostedion canadense)
• White Bass (Morone chrysops)
• Paddlefish (Polydon spathula)
• Electro-shock Sampling Performed

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Samples
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Data Preparation

• K(t) calculated from Standard data
• K(t) Area 180 Area of FAME
• Weight of 180 Weight of FAME
• FA Weight K(t) Sample Area FAME 100 .
• Sum of Sample Area of 13 FAMEs
• Degree of Saturation Calculated by adding FA
  Weight data

• n-3 FAs
• n-6 FAs

• SFAs
• MUFAs
• PUFAs

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FA Ratios(Calculated from FA Weight )

• n-3 / n-6 affects fish physiology and varies with
  season
• LNA / LA influences rate of elongation
  desaturation
• EPA / AA influences eicosanoid production
• DHA / AA effected by ratios of LNA / LA

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Chemometric Analyses Statistical Analysis System
(SAS) Software

• Analysis of Variance (ANOVA)
• Principal Component Analyses (PCA)
• Cluster Analyses
• Correlations

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ANOVA (SAS PROC GLM)

• Determined significant differences between means
  at a 0.05 level
• Transformed data 57.295779 (Arcsine v FA Weight
  / 100 )
• Used Duncan multiple-range test for unequal
  sample sizes

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PCA (SAS PROC PRINCOMP)

• Transforms possibly correlated variables into
  less uncorrelated variables called principle
  components (PCs)
• Eigen analysis solves for eigenvalues and
  eigenvectors of a square symmetric matrix with
  sums of squares and cross products
• Goal
• reduce dimensionality of data set
• identify new meaningful underlying variables

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Cluster Analyses (SAS PROC CLUSTER)

• Correlation coefficients used to group categories
  from most similar to least similar
• Used first three PCs
• Dendrograms represent output from cluster
  analysis (SAS PROC TREE)

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Correlation Coefficients (r)(SAS PROC CORR REG)

• CORR used to determine linear correlations
• REG used to determine significance
• a 0.05
• a 0.10

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Chemometric Analyses

• Significant Factors
• Species (3 Species Sampled)
• Season (3 Seasons Sampled)
• Location (6 Locations Sampled)
• Control Significant Factors
• Must hold all significant factors constant
  except the one being analyzed

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Age

• This Research Group Concluded
• Lean fish demonstrated no difference in white
  muscle lipid levels with age
• (sauger, bass, crappie and walleye)
• Fat fish demonstrated increased lipid levels in
  white muscle with increased age (channel catfish,
  smallmouth buffalo)
• Paddlefish were believed to increase lipid levels
  with age, but only young of the year samples were
  provided.

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ANOVA of FAs Among Species




Within a FA, Means with the same letter are not
significantly different (a 0.05).
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PCA of FAs Comparing Species
Sauger (S) White Bass (W) Paddlefish (P)
PC2 (10.65)
PC1 (73.45)
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ANOVA of FA Categories Among Species
Within a category, means with the same letter are
not significantly different (a 0.05).
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ANOVA of FAs in White Bass Comparing Sampling
Month
Within a FA, Means with no or the same letter are
not significantly different (a 0.05).
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PCA of FAs Comparing Sampling Month in White Bass
June (J) August (A) October (O)
PC2 (26.31)
PC1 (41.61)
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Cluster Analysis of White Bass Comparing Month
August October June
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Correlations with Julian Sampling Date
n-3 FAs in Sauger
SFAs in Sauger White Bass
MUFAs in White Bass
MUFAs in Sauger
n-6 FAs in Sauger White Bass
n-3 / n-6 FAs in Sauger White Bass
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Cluster Analysis of PCs of FAs in White Bass
Comparing Pools
Belleville Greenup Hannibal Cannelton Meld
ahl Smithland
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Correlations with Logarithm of River Mile in
Sauger (p lt 0.05)
161n-7 r 0.812
183n-3 r 0.917
182n-6 r 0.844
140 r 0.916
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Subpopulations

• FAs classified sauger and white bass into
  subpopulations at river mile 341
• Upper Subpopulation Hannibal, Belleville, and
  Greenup Pools
• Lower Subpopulation Meldahl, Cannelton and
  Smithland Pools

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PCA Comparing Upper and Lower Subpopulations of
White Bass
86.7 correctly into Lower Subpopulation
87.5 correctly into Upper Subpopulation
PC2 (24.45)
PC1 (44.28)
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PCA Comparing Upper and Lower Subpopulations of
Sauger
PC2 (20.36)
87.5 correctly into Lower Subpopulation
80 correctly into Upper Subpopulation
PC1 (43.04)
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Eigenvectors of PC1 used to Differentiate
Subpopulations
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ANOVA of Subpopulations
Within a parameter species, means with the same
letter are
not significantly different (a 0.05).
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Identification of Subpopulations Conclusions

• FAs do provide information about aquatic
  environments
• 140, 161n-7, 181n-7 and 226n-3 were the four
  most significant FAs with regard to subpopulation
• Supports use of FAs as indicators

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Conclusions

• Comprehensive Literature Review
• Provides overview of research
• Aids in identify factors effecting FAs
• FA Identity Confirmation using Two Methods
• Supported Ohio River Research
• Creation of new standard for future research
• Significance of Species, Season Location
• Determination required for analysis of FA data
• Supports continued research on Ohio River Fish

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• THE END
• of the Fish Tales

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Nutritional Effects of Lipids

• Lipids - energy source
• Required for growth, repair maintenance of
• Damaged tissue
• Normal Egg development
• Immune system

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Essentially Fatty Acids (EFAs)

• Required NOT biosynthesized
• Freshwater species generally require
• LNA (183n-3) LA (182n-6)
• Marine species generally require
• EPA (205n-3) DHA (226n-3)

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EFAs Deficient Diets

• Reduced
• Growth
• Spawning efficiency
• Hatching
• Swimming activity
• Feed efficiency
• Survival rate
• Appetite

• increased mortality
• shock syndrome
• fainting
• swimming disorders
• elevated muscle water content
• Decreased water-temperature tolerance

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Physiological Effects on FAs

• Species Age
• Sex Tissue Sampled
• Metabolism
• Lipogenic Enzymes
• Exercise Performance
• Competitive metabolism among EFAs
• Immune System Function Eicosanoids
• Cardiovascular Respiratory Function
• Reproductive Function

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Eicosanoids

• Highly active C20 PUFA derivatives
• Normal physiological process
• Regulation of physiological processes
• Inflammatory-response
• Blood clotting
• Cardiovascular tone
• Reproductive function
• Membrane fluidity

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Water Quality Effects FAs

• Organochlorine Compounds
• Positively correlated with lipids
• Mercury
• Inorganic mercury preferentially
• bound to unsaturated FAs
• Endocrine-Disruption Chemicals
• Altered lipid distribution

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Extraction of Lipids

• White muscle (1 g) homogenized (4-6
  min.) in chloroformmethanol (21, vv )
• Vacuum filtered (Coors
  60240 funnel, Whatman 1)
• Filtrate transferred NaCl solution added
  (0.125M, 20 of the filtrate volume)

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Extraction (continued)

• Stoppered cylinders refrigerated (12 to 48h)
• Na2SO4 added to lower chloroform phase (2g)
• Filtered (Coors 60240 funnel, Whatman 1)

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Hydrolysis to Fatty Acids

• Pure Lipid (5 mg) transferred chloroform
  evaporated
• NaOH added (1.5 mL, 0.5M)
• Vortexed (15 25 sec.)
• Heated (5 min. _at_ 100?C)
• Cooled (beaker tap water)

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Hydrolysis of Triacylglyceride to Fatty Acids
Triacylglyceride Glycerol Fatty Acids
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Derivatization/Esterification FA ? Fatty Acid
Methyl Ester (FAME)

• Add BF3-methanol (2mL)
• Vortexed (15-25 sec.)
• Heated (20 min., 100?C)

FA FAME
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Derivatization (continued)

• Cooled (37?C water bath )
• Isooctane added (1mL)
• Vortexed (15-25 seconds)
• NaCl added (3 mL, saturated)
• Inverted repeatedly (1 minute)

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Derivatization (continued)

• Centrifuged (1300 rpm, 2 minutes)
• Upper isooctane phase transferred to tube
  containing anhydrous Na2SO4 (2mm layer)
• Shaken stood (20 min.)
• Isooctane transferred to vials for GC analysis

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FID Overview
The effluent from the column is mixed with
hydrogen and air, and ignited. Organic compounds
burning in the flame produce ions and electrons,
which can conduct electricity through the flame.
A large electrical potential is applied at the
burner tip, and a collector electrode is located
above the flame. The current resulting from the
pyrolysis of any organic compounds is
measured. The FID is a useful general detector
for the analysis of organic compounds it has
high sensitivity, a large linear response range,
and low noise. It is also robust and easy to use,
but unfortunately, it destroys the sample.
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Species Selection Sauger (Stizostedion
canadense) and White Bass (Morone chrysops)

• Large river Migratory species
• Live gt 2 years
• Make distinctive spawning runs
• Spawn within fairly short period
• Sought after by anglers

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Paddlefish (Polydon spathula)

• Paddlefish in sturgeon family and eggs may be
  consumed as caviar
• Oldest surviving animal species in North America
  older than dinosaurs
• Only 9 young of year samples received

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Sauger

• Kingdom Animalia
• Phylum Chordata
• Subphylum Vertebrata
• Class Actinopterygii
• Order Perciformes
• Suborder Percoidei
• Family Percidae
• Genus Stizostedion (pikeperches)
• Species Stizostedion canadense

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White Bass

•      Kingdom Animalia
•      Phylum Chordata
•      Subphylum Vertebrata
•      Class Actinopterygii
•      Order Perciformes
•      Suborder Percoidei
•      Family Moronidae
•      Genus Morone (Striped Bass)
• Species Morone chrysops

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Paddlefish

• Kingdom Animalia
• Phylum Chordata
• Subphylum Vertebrata
• Class Actinopterygii
• Order Acipenseriformes
• Family Polyodontidae
• Genus Polyodon (paddlefishes) ,
• Species Polyodon spathula (American paddlefish or
  paddlefish)

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Electro-shock Sampling

• Pulsed DC boat-mounted (4-6A, 60Hz)
• Performed by WV, KY, IN, IL state agencies
• Boat length 18 20 ft.
• Smith-Root GPP 9.0 unit
• Dropper length 5 7 ft with 1-2 feet in water

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Significant Correlations (r) with Julian Sampling
Date in both Sauger and White Bass (p lt 0.05)
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Correlations between River Mile and FAs
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ANOVA of FAs in White Bass Comparing
Subpopulations




Within a FA, Means with no or the same letters
are not significantly different (a 0.05).
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ANOVA Relative Weight Comparing Pools
Means with the same letter are not significantly
different (a 0.05).
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Benefits Multi-faceted

• Ohio River Fish Management Teams
• Future Research
• Ohio River Species
• Using FAs as Bioindicators
• Fish Fish Oil Industries
• People who eat fish