Dietary Lipid Utilization by Haddock (Melanogrammus aeglefinus) Santosh P. Lall & Dominic A. Nanton National Research Council of Canada Halifax, Canada vis, California ne 23, 2003
Body Components of Wild Haddock Head, 21.8% 37.4% Fillet (skinned) Gut, 7.5% Liver, 5.2% Skin, 3.1% Fins, 10.4% Vertebrae, 14.6%
Haddock Lipid Nutrition Cultured gadoids have abnormally high liver lipid deposition when fed moderate level of lipid in their diets Fish with fatty livers are: inefficiently using the dietary energy, may have lower somatic growth rates and increased susceptibility to stress and infectious disease. Studies designed to provide information on retention, transport and catabolism of lipid in haddock.
Fatty Liver Syndrome in juvenile haddock HSI HSI = 12.3% = 12.3% HSI HSI = 18.4% = 18.4%
Liver Function of Haddock Series of liver function tests performed on plasma of cultured haddock grouped on basis of their HSI Cell Integrity Alanine aminotransferase, aspartate aminotransferase, sorbitol dehydrogenase Cholestasis Alkaline phosphatase, gamma-glutamyl transferase Liver Metabolism: Synthesis Total protein, albumin Liver Metabolism: Clearance Total bilirubin
Summary No clear evidence or trend of hepatic dysfunction in haddock with individual HSI ranging from 10.6 to 19.6% or differences between groups with an average HSI of 11.1, 13.0 and 17.3%.
Lipid Deposition in Haddock Determine the effect of dietary lipid levels on growth and biochemical composition of juvenile haddock Biological criteria: Growth, feed efficiency and hepatosomatic index Biochemical criteria: Lipid and fatty acid composition of liver and muscle (neutral and polar lipids)
Proximate composition of the experimental diets Composition Dietary lipid levels (%) (g/100 g) 12 15 18 21 Moisture 13.34 12.96 12.85 12.71 Protein 54.34 54.78 54.83 55.06 Lipid 13.78 16.13 18.89 21.50 Fiber 0.87 0.76 0.60 0.47 Ash 6.90 6.94 6.95 6.90
Growth and feed utilization of haddock fed graded levels of lipid for 9 weeks Lipid level Wt. gain SGR Feed:gain (%DM) (g/fish) (%) Ratio 1 14 30.6 ns 2.7 ns 0.69 ns 16 32.3 2.8 0.66 19 32.7 2.8 0.68 22 33.0 2.8 0.65 Average initial wet weight was 6.9g 1 Feed intake (expressed as dry matter)/wet wt. gain
Effect of dietary lipid level on hepatosomatic index in juvenile haddock Hepatosomatic Index (%) 14 12 10 8 6 4 2 10.84ab 11.30bc 9.78 a 12.10 c 0 14 16 19 22 Dietary Lipid (% DM)
Lipid composition of haddock fed graded levels of lipid Dietary Liver Muscle Lipid (% DM) Lipid (%) Lipid (%) 14 16 19 22 63.16 ± 2.41 a 64.32 ± 1.98 a 67.02 ± 1.30 ab 68.95 ± 2.42 b 0.95 ± 0.03 ns 1.05 ± 0.09 1.05 ± 0.10 1.01 ± 0.04
Conclusions Increasing dietary lipid from 14 to 22% (dry wt.) did not significantly affect growth and feed efficiency. HSI and liver lipid significantly increased with dietary lipid (14 to 22% dry wt.) whereas muscle lipid remained low at appr. 1%. A dietary lipid level of 14% or less is recommended for juvenile haddock.
Lipid Transport in Haddock Objective: Evaluate distribution and composition of serum lipoproteins in juvenile haddock No research on lipoprotein profile of haddock (gadoids) Information on level and lipid composition of VLDL will increase our understanding of lipid transport out of the liver in haddock.
Amphipathic Alphahelical Proteins (Apo E and Apo C) Apo B Neutral Lipid Core (Cholesterol Esters, Triacylglycerols) Monolayer Surface OH Phospholipids Free Cholesterol Schematic Diagram of VLDL
Lipoprotein Separation Volume (ml) Density (g/ ml) 2.2 Top 1.006 2.5 1.019 2.0 1.063 3.0 1.210 (serum sample) 2.0 Bottom 1.240 - KBr density gradient prepared in an ultracentrifugation tube. - Serum adjusted to final density of 1.210 g/ ml in 3.0 mls using KBr solution.
Centrifugation, Fractionation, Lipid Analysis Centrifuged at 40,000 rpm for 44 h at 15ºC. Successive fractions of 0.4 mls aliquotted from meniscus downward with a pipette for the entire density gradient. Individual fractions derivatized, extracted and injected on GLC using lipid profiling technique of Kuksis et al. (1978).
CHOL PL CE TAG C- 30 (STD) C- 32 C- 34 C- 36 C- 38 C- 40 C- 42 & C- 43 C- 44 C- 45 C- 47 C- 48 C- 49 & C- 50 C- 52 C- 54 C- 56 C- 58 C- 60 C- 62 OVERLAP OVERLAP C- 27 170 250 300 350 Temperature (ºC) Total lipid profile from the plasma of haddock analyzed by the GC method of Kuksis et al., 1978
Composition of serum (mg/dl) 600 500 400 300 200 100 Composition of Haddock Serum triacylglycerol phospholipid cholesterol ester free cholesterol protein 0 Fraction No. 1 2 3 4 5 6 7 8 9 101112131415161718192021222324 VLDL (d<1.017) LDL (1.056) HDL (1.097) 1.009 Density (g/ml) 1.225
900 Whole Serum Lipid Composition of Haddock 800 Serum lipid (mg/dl) 700 600 500 400 300 200 100 0 Free cholesterol Cholesterol ester Phospholipid Triacylglycerol
60 Fatty acid composition of whole serum, serum PL, HDL and muscle in haddock 50 Fatty Acid (%) 40 30 20 SAT MONO PUFA 10 0 Whole serum Serum PL HDL Muscle (n=6) (n=6) (n=3) (n=5)
50 Fatty acid class composition of liver, serum triacylglycerol and VLDL in haddock Fatty Acid (%) 45 40 35 30 25 20 15 10 5 SAT MONO PUFA 0 Liver Serum TAG VLDL (n=5) (n=6) (n=3)
60 Fatty acid composition of diet, liver, VLDL and muscle in haddock 50 Fatty Acid (%) 40 30 20 SAT MONO PUFA 10 0 Diet Liver VLDL Muscle (n=2) (n=5) (n=3) (n=5)
Exogenous Pathway Dietary Lipid Bile Components Endogenous Pathway?? LDL Intestine FA Fatty Liver Extra- Hepatic Tissues? Chylo LPL Remnant I B TG A-I B C VLDL B LPL IDL? LCAT? HDL TG A-I A-II Exogenous and endogenous lipid transport pathways in fish
Summary HDL predominant lipoprotein with relatively low amounts of VLDL (<50 mg/dl) in haddock serum Total serum lipid comprised of 58% phospholipid and only 15% triacylglycerol Haddock may have limited ability to increase transport of deposited fat from liver to muscle for storage as dietary lipid level increased
Lipid Catabolism in Haddock Red muscle and heart have higher fatty acid ß-oxidation activities than white muscle and liver in fish. Two organelles for ß-oxidation: mitochondria and peroxisomes. Peroxisomes more important than mitochondria for ß- oxidation in liver (30-100% of total) compared to muscle of fish. First study to measure lipid catabolism in tissues of a gadoid fish (liver is major lipid storage organ).
Introduction 1 Does site of lipid storage (liver) affect activities and pattern of ß-oxidation in the tissues of haddock? 2 Does increasing dietary lipid level increase ß-oxidation activities in the tissues of haddock? Measured lipid composition and ß-oxidation activities of tissues (red muscle, white muscle and liver) in juvenile haddock fed 12, 18 and 24% lipid
Fatty Acid 1 Cytosol Fatty acyl-coa Peroxisom l coa synthetase itine palmitoyltransferase I l carnitine translocase itine palmitoyltransferase II itine acetyl transferase Fatty acyl-coa Acetyl CoA B-oxidation 2 Acetyl CoA Fatty acylcarnitine 3 Fatty acylcarnitine 4 Fatty acyl-coa 5 Acetylcarnitine Mitochondrion H 2 O 2 B-oxidation Acetyl CoA TCA Cycle im om CO 2 +ATP
Mitochondrial ß-oxidation CH 3 (CH 2 CH 2 ) n CH 2 CH 2 CO-S-CoA Peroxisomal ß-oxidation CH 3 (CH 2 CH 2 ) n CH 2 CH 2 CO-S-CoA FAD O 2 Acyl-CoA dehydrogenase Acyl-CoA oxidase FADH 2 H 2 O 2 Catalase H 2 O + 1/2O 2 CH 3 (CH 2 CH 2 ) n CH=CH CO-S-CoA CH 3 (CH 2 CH 2 ) n CH=CH CO-S-CoA H 2 O Enoyl-CoA hydratase Bi(Tri)functional enzyme H 2 O CH 3 (CH 2 CH 2 ) n CHCH 2 CO-S-CoA NAD + NADH OH 2-Hydroxyacyl-CoA dehydrogenase CH 3 (CH 2 CH 2 ) n CHCH 2 CO-S-CoA CoA-SH O Thiolase CH 3 (CH 2 CH 2 ) n CHCH 2 CO-S-CoA Bi(Tri)functional enzyme NAD + NADH CH 3 (CH 2 CH 2 ) n CHCH 2 CO-S-CoA Thiolase OH O CoA-SH CH 3 (CH 2 CH 2 ) n CO-S-CoA + CH 3 CO-S-CoA CH 3 (CH 2 CH 2 ) n CO-S-CoA + CH 3 CO-S-CoA
18 17 Hepatosomatic index of juvenile haddock fed three levels of dietary lipid (12, 18 and 24%). c Hepatosomatic index (%) 16 15 14 13 12 a b 11 10 9 12% 18% 24%
90 80 a b Lipid composition of tissues in juvenile haddock fed graded levels of lipid (12, 18 and 24%). b 70 60 50 40 12% 18% 24% 30 20 10 0 Average =1.9% Average =0.8% Average =2.5% Liver Red muscle White muscle Heart
70 60 50 Fatty acids in tissues of haddock fed 18% lipid Saturated Monounsaturated Polyunsaturated 40 30 20 10 0 Diet (n=2) Liver (n=4) Red muscle (n=5) White muscle (n=5) Heart (n=5)
red muscle white muscle mpling of Red and White Muscle from Skinned Haddock Fillet
oxidation of palmitoyl- A per mg protein 20 C in juvenile ddock fed graded vels of lipid (12, 18 and %) Activity (nmol/ min/ mg protein) Activity (nmol/ min/ mg protein) 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0.00 a a a a a 12% a 18% 24% b b b Mitochondria b b b Peroxisomal c c c Liver (n=5) Red muscle (n=4) White muscle (n=4)
oxidation of palmitoyl- A per g wet tissue at C in juvenile haddock d graded levels of lipid 2, 18 and 24%). Activity (nmol/ min/ g wet tissue) Activity (nmol/ min/ g wet tissue) 10.0 8.0 6.0 4.0 2.0 0.0 2.5 2.0 1.5 1.0 0.5 0.0 a ab b a c a 12% 18% 24% d a cd Mitochondria b b Peroxisomal e e e Liver (n=5) Red muscle (n=4) White muscle (n
Total ß-oxidation of palmitoyl-coa per g fish at 20 C in liver of juvenile haddock fed graded levels of lipid (12, 18 and 24%). Activity (nmol/ min/ g fish) 0.30 0.25 0.20 0.15 0.10 a a a 0.05 0.00 12% (n=5) 18% (n=5) 24% (n=5)
Summary No significant increase of ß-oxidation activities in tissues of haddock fed 12 to 24% lipid Mitochondrial (>90%) ß-oxidation was dominant in the muscle and peroxisomal (100%) in the liver Red muscle possessed the highest specific activity for ß- oxidation White muscle and liver had similar but lower activities on a per g tissue basis White muscle comprises over 50% of the body wt. and is the most important tissue for overall fatty acid catabolism
Overall Conclusions Results from these studies on lipid deposition, transport and catabolism in haddock indicate that: The transport of lipid from the predominant storage (liver) to catabolic (muscle) site is low in haddock. This suggests the underlying cause of fatty liver in cultured gadoid fish.
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