I
Page 1 of 1
From: To: Sent: Subject:
OAO Tuesday, October 17,2000 4:15 PM
WWW ILL Request
[email protected] (OAO) sent the following ILL request:
................................................................
Author: R. E. Olsen, R. Myklebust, E. Ring0 and T. M. Mayhew, Book Title: Fish Physiology and Biochemistry, Journal Article: The influences of dietary linseed oil and saturated fatty acids on caecal enterocytes in Arctic char (Salvelinus alpinus L.): a quantitative ultrastructural study. Year: 2000 Volume: 22 Pages: 207-2 16 Location: Rush: on Needed By: User Name: OAO E-Mail address:
[email protected] Phone #: 17-10-00 Server Protocol: HTTP/l. 1 Remote Host: (200.10.149.1 1)
11/5/00
Fish Ph\siolog\ und Biochetnistn 22: 207-2 16. 2000. 0 2000 K l u w r Arudemic Publishers. P rirzted in rhe Netherlands.
207
The influences of dietary linseed oil and saturated fatty acids on caecal enterocytes in Arctic char (Sulvelinus alpinus L.): a quantitative ultrastructural study R. E. Olsen' , R. Myklebust', E. Ring$ and T. M. Mayhew4
'
~
~
Departineiit ?fFisheries. Finnmark College. Follurnsvei, N-YSOO Aha. N o n t q . : Present address: Institute c?f Marine Research. Matre Aquaculture Research Station, N-5984 Matrpdal. Nom.ay (Phone: +47 56366040; Fax: +47 56366143: E-mail:
[email protected]): 'Department of M@nholog~/Departmentof Electmn Microscopy? 7 F a d ? of Medicine. Uniiiersir?. of TromsB. N-9037 Tromso. Norway; - Department ?f Arctic Veterinan Medicine, The Norwegian School of Veterinag Science, Stakkevollveien 23 b. N-9292 Tromsg, Norway: 4School of Biomedical Sciences. Queen 'sMedical Centre, Universih of Nottingham, Nottingham NG7 2UH, UK Accepted: 12 Januac 2OOo
KeJ.wards: epithelial damage. fish. intestine. lipid digestibility. lipid droplets. lipid nutrition. lipoprotein synthesis, ultrastructure
Abstract Arctic char (Sah~e1inu.salpinus L.) were fed over a three-week period with a commercial diet or one of seven casein-based diets. The latter were either lipid-free or supplemented with 50-200 g linseed oil kg-' diet by dry weight. 160 g linseed oil and 40 g 14:O or 160 g linseed oil and 40 g 16:O. Three fish having filled guts were sampled from each dietary group and analysed for ultrastructural changes in the pyloric caecum. Increasing the dietary lipid level increased the accumulation of lipid droplets in columnar absorptive enterocytes ' in fish fed a diet of 200 g from about 9% of epithelial volume in fish fed a diet of 50 g linseed oil, to almost 61% linseed oil. Replacing linseed oil in the diet with 14:O ( 160 g linseed oil 40 g 14:O kg-' diet) appeared to produce a bmaller lipid loading (roughly 53% ) but the difference was not statistically significant. However. replacing 40 g linseed oil with 40 g of 16:O in the diet decreased lipid loading significantly to just under 10%.Epithelial damage to enterocytes was assessed using a ranking scale based on ultrastructural signs of cell and organelle swelling and degeneration. The extent of damage closely followed the level of lipid loading. being lowest in fish fed the lipid-free or lo%,-lipid(damage index 0.07-0.13) diets. and highest in char maintained on a diet containing 200 g linseed oil (index 1.411. Replacing linseed oil with 14:O (160 g linseed oil + 40 g 14:O) appeared to reduce the damage index to 0.77 but this was not significant. However. a significant reduction of the damage index to 0.27 was observed when linseed oil was replaced by 16:O. We conclude that higher dietary linseed oil promotes lipid droplet accumulation in enterocytes. The droplets are probably related to the amount of polyunsaturated fatty acids in the lipid. lntracellular droplet formation and cellular damage are both reduced by adding saturated fatty acids to the diet. This could be related to disruptions in the lipoprotein assembly rate. The cellular damages observed with high lipid diets are likely to be pathological and may lead to intestinal malfunction and represent a major infection route for pathogenic bacteria.
+
Introduction Free lipid are droplets sometimes observed accumulating in fish enterocytes during nutrient absorption (Bergot and Flechon 1970a,b; Noaillac-Depeyre and Gas 1974. 1976. 1979; Stroband and Debets 1978;
Sire and Vernier 198 1 : Deplano et al. 199I : Sarasquete et al. 1995; Fontagne et al. 1998). Although cells may become loaded with these droplets, they have rarely been considered as having harmful effects on cells. Recently. however, Olsen et al. (1999) showed that
208 feeding Arctic char a diet containing 150 g linseed oil (kg-' led to extensive damage to enterocytes that seemed to be caused by accumulation of lipid-laden droplets. Such damage was probably pathological and possibly detrimental to fish health. The rationale for the formation of lipid droplets is somewhat obscure. They are not part of the endoplasmic reticulum or the Golgi apparatus of the cells. In many cases they do not appear to be enclosed in any membrane. This has led to the suggestion that lipid droplets are temporary storage forms of lipid in cases when the rate of lipid absorption exceeds the rate of lipoprotein synthesis (Iwai 1969: Bergot and FlCchon 1970b: Noaillac-Depeyre and Gas 1974: Stroband and Dabrowski 1979: Watanabe and Sawada 1985 ), However. there are no extensive studies showing a dose-response relationship between dietary lipid load and intracellular droplet content. Another factor that can influence the formation of lipid droplets in enterocytes is the nature of the dietary fatty acids. It is generally accepted that higher levels of unsaturation increase lymph lipoprotein size in mammals (Degrace et al. l996a.b; Sakr et al. 1997). Feeding rainbow trout a balanced diet stimulates synthesis of very low-density lipoproteins and formation of small chylomicrons (Sire et al. 1981). A diet with excess lipids comprising a large portion of polyunsaturated fatty acids (PUFA) results in formation.of large chylomicron-like particles and the temporary appearance of lipid droplets (Sire and Vernier 1981). The relationship between lipid load and unsaturation requires further investigation. The present work aims to study the effects of lipid load and unsaturation on lipid droplet formation and cellular damage in the caecal enterocytes of Arctic char. The oil chosen for this study was linseed oil. It is one of the few plant oils available that contain mainly n-3 PUFA which is the major essential fatty acid for salmonids. *.-
Materials and methods
Purified linseed oil was purchased from Roth ( k l sruhe. Germany) while tkmyristin (tri-14:O) and tripalmitin (tri- I6:O) were from Sigma (St. Louis, USA ). Astaxanthin (Carophyl pink 80 g kg-') and tryptophan were obtained from Hoffman-La-Roche (Hvidovre. Denmark). Gelatine and potassium sorbate were purchased from Merck (Darmstadt. Germany 1.
betaine from Janssen Chemica (Geel, Belgium). and vitamin mixture 76 from ICN Biomedicals (Cleveland. USA). The mineral mixture was kindly donated by Felleskjapet Ltd (Dirdal. Norway) and was identical to the mixture used in their commercial tish diets. All other chemicals and dietary ingredients were purchased from Sigma (St. Louis. USA). Fish
Arctic char (Solvelinus d p i n u s L. 1 of the anadromous Hals River strain (70"N. 23''E) were reared in freshwater at Talvik Research Station. FE.AS. Northern Norway under ambient light and temperature conditions until they attained an average weight of 250 g. In May. the fish were divided into eight sroups (each containing 20 fish) and transferred to I20 1 PVC selfcleaning tanks supplied with aerated fresh water at a constant temperature of 9 'C. The rearing groups were allowed to adapt over a two-week period to the new environmental conditions and experimental diets. Food was supplied in excess ( 2 % ) every morning between 09.00 h and 10.00 h using disc feeders. After adaptation. fish were fed the test diets for three weeks. One group of experimental fish was maintained on the commercial diet. Diets
The dietary compositions are given in Table I . Diets were prepared by first mixing the dry ingredients (casein, dextrin and premix 1 ) in a commercial blender. Premix I was based on vitamin mixture 76 further supplemented with purified vitamins. For simplicity. the vitamins in Table 1 are given as total vitamin content of the diets. Premix 2 (a-tocopheryl acetate and the antioxidant ethoxyquin) was dissolved in the lipid source and added to the blender. followed by the addition of warm water (600 g I-' ) into which premix 3 and gelatine were dissolved. The mixture was thoroughly mixed. pelleted through a 5 mm die and sieved to obtain uniform particle size. All diets were placed ik plastic bags containing a one-week supply. stored at -20'C and put fresh onto the disc feeders (moist pellets) each day. Linseed oil was added as lipid source from 50 to 200 g kg-l diet by dry weight. except for one diet where no linseed oil was added. and two diets where 40 g of linseed oil was replaced by either 40 l4:O or 40 g 1 6 9 in addition to 160 g linseed oil (Table I ) . A standard commercial diet containing 250 g kg-l marine lipid and protein was used as control. Total
209 Table 1 Composition of the expenmental diets (ingredient contents given in g kgof d c weight I uced in the pre\ent ctud!
Premix 1 . 2 and 3 Gelatin Dextnn Casein Linseed oil 1l : o
I
2
3
1
5
6
7
12 I5
42 I5
12
42 15
42 IS
12 I5
150 M3
150
150
593
593
12 15 150 593
I50
200
160
160
150 793
0
15
I50
I50
73.:
693
50
__
." 100
* Comm.
40
-".
16:O
8
'
40
Premix 1 : (mg k g - ' h j d n weight): Thiamin 10. riboflavin 20. biotin 1. pyndoxin 10. niacin 150. pantothenic acid 40. folic acid 5. vitamin B I Z0.02. vitamin C 100. vitamin A 3115 IU. vitamin D So00 IU. vitamin K 10. p-aminobenzoic acid 200. inositol 300. phenylalanine 2000. tryptophan 2500. glycine 2000. mineral mixture 3OOO. astaxanthin (Carophyl pink) 20oO. sucrose (carrier for vitamin mixture 76) 12161. Premix 2 . imp hg-] b! d n weight,: a-tocopheryl acetate 310. ethoxyquin 200. Premix 3: cmg kg-I b! dry weight): Betaine 3OOo. potassium sorbate IooOO. citnc acid 1 0 . choline chloride 3OOO. *An extruded commercial diet was used as control. It contained 2.50 g kg-I (by analysis) marine lipid of diet by d v weight.
lipid fatty acid Compositions of the diets are given in Table 2.
Fat6 acid analysis Total lipid was extracted from the diets according to the method of Folch et al. (1957). After evaporation to dryness in ~ ~ u c uato room temperature, total lipid was redissolved in chloroform:methanol(2: 1 v/v) and stored under nitrogen at -20 'C prior to analysis. Total lipid methyl esters were prepared by sulphuric acid-catalysed transmethylation according to Christie ( 1987) using 19:O as an internal standard. The methyl esters were separated on a Dani 8500 gas-liquid chromatograph (Dani Strumentazione Analitica Spa. Milan. Italy) equipped Q.ith a WCOT fused silica (SO m x 0.25 mm ID) capillary column with a tailor-made CP-SIL 88 coating and 2.5 m retention gap. methyl deactivated (Chrompack International BV. Middelburg. the Netherlands). Peaks were quantified on a HP 3365 series I1 Chemstation. The temperature program was initiated at 60 "C and maintained for 1.7 min followed b), an increase of 95 'C min- to 165 'C. which was maintained for S min. The temperature was then increased b) 1 S "C min-' to 240 'C and maintained for 17.5 min.
'
Tissue processing f o r microscop!
For electron microscopical examination of intestines. three fish from each hetary regimen were sacrificed
and guts dissected according to the method of Olsen et al. (1999). above. In each ease, only fish containing digesta throughout the whole length of the intestine were used. Sections from the pyloric caeca were dissected out and immediately fixed in McDowell's fixative (McDowell and Trump 1976) and dissected into smaller pieces suitable for transmission electron microscopy (TEM). Pieces were then washed in phosphate buffer (300-320 mOsm. pH 7.4). postfixed for 1.5 h in osmium tetroxide. iewashed in phosphate buffer and stained en bloc in 9 4 aqueous uranyl acetate. After dehydration in a graded series of ethanol concentrations. the specimens were embedded in EpodAraldite via propylene oxide. U1trathin sections were contrasted with uranyl acetate and lead citrate and examined in a JEOL 1010 transmission electron microscope at an accelerating voltage of 80 kV. Fields of view were recorded at a nominal instrument magnification of x 1 500 and printed as micrographs at a final magnification of x3750 calibrated with the aid of a carbon grating replica external standard. After fixation and dehydration some of the specimen were prepared for scanning electron microscopy (SEM j. The specimen were critical-point dried. using CO,. mounted on aluminium stubs. coated with gold and viewed on a JEOL. JSM 5300 scanning electron microscope.
I
210 Table 2. Content of the major fatty acids (mol%) of total lipid extracted from the diets used in the present study
Dirt I* 3
7
I6:O 16:I n-7
10.5 tr
I n:o 18: I n-9
4.0 19.8 20.3 4S.2 nd nd nd
I8:ln-6 18:3n-3/10: In-9 22:ln-I I 2051-3 22:6n-3 *Not a n d y e d . nd. not detected. tr. trace only (
