Effects of highly purified sardine proteins on lipid peroxidation and reverse cholesterol transport in rats fed a cholesterol-rich diet

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Effects of highly purified sardine proteins on lipid peroxidation and reverse cholesterol transport in rats fed a cholesterol-rich diet Sabrine Louala, Sherazed Hamza-Reguig, Aicha Benyahia-Mostefaoui, Ahmed Boualga, Myriem Y. Lamri-Senhadji * Laboratoire de Nutrition Clinique et Me´tabolique (LNCM), Faculte´ des Sciences, De´partement de Biologie, Universite´ d’Oran, 31000 Oran, Algeria

A R T I C L E I N F O

A B S T R A C T

Article history:

The effect of highly purified sardine proteins was compared with that of casein on serum

Received 29 January 2011

and lipoproteins lipid peroxidation and reverse cholesterol transport. Lecithin: cholesterol

Received in revised form

acyltransferase (LCAT) activity, high density lipoproteins (HDL2 and HDL3) composition and

20 May 2011

serum lipid and lipoproteins peroxidation were determined in rats fed a cholesterol-rich

Accepted 5 July 2011

diet. Hypercholesterolemic rats were divided into two groups fed diets enriched with cho-

Available online 12 August 2011

lesterol and containing 20% of highly purified sardine proteins (SPc) or casein (CASc) for 28 days. A control group was fed a standard diet (CAS). Compared with CAS and CASc,

Keywords: Rat

the thiobarbituric acid reactive substances (TBARS) concentrations of low density lipoprotein (LDL)–HDL1 in SPc were 3.5- and 1.7-fold higher compared with casein diets. TBARS in

Sardine

HDL2 and HDL3 were, respectively, 2.3- and 1.6-fold lower in SPc compared with CASc. In

LCAT Lipid peroxidation Lipoproteins Cholesterol

SPc group, LCAT activity was higher compared to CASc and CAS (P < 0.05). Purified sardine proteins had no beneficial effects on LDL-cholesterol and lipid peroxidation. However, they reduced HDL oxidation and improved reverse cholesterol transport, in the hypercholesterolemic rat. Ó 2011 Elsevier Ltd. All rights reserved.

1.

Introduction

Increased plasma low density lipoprotein (LDL) concentration is associated with the susceptibility to developing atherosclerosis (Penumathsa et al., 2007). Although the precise mechanism of atherogenesis still needs further investigations, oxidative modification of LDL is considered to be an essential process in the activation of the inflammatory pathway,

leading to atheromatous plaque formation in the intimal layer of the artery (Witztum & Steinberg, 1991). In contrast to the adverse effects of elevation of LDL, the concentration of high density lipoprotein (HDL) is inversely correlated with atherosclerosis development, apart from its role in reverse cholesterol transport (Khera & Rader, 2010) and as an antioxidant inhibits the oxidative modification of LDL (McPherson, Young, & McEneny, 2007).

* Corresponding author: Tel.: +213 41581944; fax: +213 41560601. E-mail address: [email protected] (M.Y. Lamri-Senhadji). Abbreviations: CAS, rats fed casein diet; CASc, hypercholesterolemic rats fed casein diet containing cholesterol; HDL, high density lipoprotein; LCAT, lecithin:cholesterol acyltransferase; LDL, low density lipoprotein; MUFA, monounsaturated fatty acid; SPc, hypercholesterolemic rats fed sardine protein diet containing cholesterol; PUFA, polyunsaturated fatty acid; SFA, saturated fatty acid; TBARS, thiobarbituric acid reactive substance; TC, total cholesterol; TG, triacylglycerol 1756-4646/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jff.2011.07.002

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Lecithin:cholesterol acyltransferase (LCAT, EC 2.3.1.43) is a key enzyme located on the surface of serum HDL involved in transesterification of free cholesterol to cholesteryl ester. This enzyme is synthesised predominantly by liver and requires, for maximal activity, apolipoprotein (Apo) A1 an activating cofactor which is found in plasma associated mainly with HDL. Recent investigations have demonstrated that the HDLassociated enzyme LCAT may play a significant role in lipoprotein modification and hence atherogenesis (Dullaart et al., 2010). Several models have been used for studying cholesterol and atherogenesis, but no single one is considered perfect for extrapolating results to humans. Rats are most animals used in cholesterol metabolism studies and one of the most often used to study the cholesterolemic effect of proteins (Chiang, Chen, & Huang, 1998). There have been attempts to induce hypercholesterolemia in rat, cholesterol-rich diets with or without cholic acid (Lichtman et al., 1999) have been used; the level of cholesterol varies substantially as well. In our laboratory we have induced hypercholesterolemia in rats with diets enriched with cholesterol (Bouderbala et al., 2009). However, lipid metabolism of the normal rat is primarily based on HDL, rather than on LDL, as in humans, possibly contributing to atherogenesis resistance (Russell & Proctor, 2006). Fish is one of the main components of a healthy diet, and many epidemiologic studies and clinical trials have indicated its beneficial effects in incidence of coronary diseases, by decreasing CHD mortality risk. This inverse association has been attributed to the hypotriglyceridemic, anti-inflammatory and hypothrombogenic effects as the positive impact on the endothelial function of x-3 polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) present in fish oil (Harris, 1997). The protein which is another active component of fish could influence lipid metabolism and may have a role as a cardioprotective nutrient. In the rat, fish protein can produce variable effects on serum total cholesterol concentrations and lipoprotein cholesterol distribution, depending in part on the amount and origin of the dietary lipid with which it is combined (Demonty, Deshaies, Lamarche, & Jacques, 2003; Shukla et al., 2006). Cod protein was shown to decrease plasma triacylglycerol (TG) and cholesterol concentrations compared with casein (Demonty et al., 2003). Similar results have been shown in Sprague–Dawley rat fed whiting protein (Shukla et al., 2006). Some studies suggested that specific amino acids could be responsible for these effects (Kritchevsky, Tepper, Czarnecki, & Klurfeld, 1982; Lavigne, Marette, & Jacques, 2000). However, little is known about the effects of sardine proteins on experimental hypercholesterolemia induced in the rat. Therefore, the aim of this study was to determine the effects of highly purified sardine proteins compared to casein, on lipoproteins lipid peroxidation and reverse cholesterol transport in rats fed a high-cholesterol diet. In this investigation sardine proteins were selected because this fish is highly consumed in the Mediterranean area.

2.

Materials and methods

2.1.

Sardine proteins purification process

Fish protein was isolated from fresh sardine (Sardina pilchardus) obtained from a local fish market (Oran, Algeria).

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Sardine proteins were obtained according to the method of Guillaume, Kaushik, Bergot, and Me´ttailler (1999). The general principle consists in separating water and oil from the dry matter. Head scales, guts and backbone were removed from fresh sardine and fillets were collected and heated in an oven at 80–85 °C (Tau Steril Snc, Fino Mornasco, Italy) for 20 min. At this level, the first separation takes place between a solid phase (coagulated proteins) and a liquid phase (water and oil). A pressing completed this operation; the cake obtained was crumbled and dried at 40–45 °C for 24 h and ground in a grinder (IKA, model A116, Hamburg, Germany). The press water was elutriated and centrifuged at 3000g (Eppendorf, Centrifuge 5702, Hamburg, Germany) to separate oil and the soluble that contained the remained proteins was reincorporated with the cake. After crushing, to remove the lipids and other organic solvent-soluble elements, the sardine proteins were washed with hexane (Biochem Chemopharma, Montre´al, Quebec, Canada) in an extractor of lipids (Soxhlet, Labo-Tech LT.6, Muttenz, Switzerland) for 5 h at 50 °C. SP concentrations were estimated by their nitrogen contents, using mineralization followed by coloration with Nessler’s reactive agent and ammonium nitrate as a standard. Then the values were multiplied by 6.25 to obtain protein content which represented 95% in sardine proteins. The characterization of the purified SP (amino acids expressed in g/kg of protein) (Garcia-Arias, Alvarez Pontes, Garcia-Linares, GarciaFernandez, & Sanchez-Muniz, 2003) is shown in Table 1. The protein content of casein (Prolabo, Fontenay sous bois, France) was 96%.

2.2.

Animals and diets

Male Wistar rats (Iffa Credo, l’Arbresle, Lyon, France), weighing 80 ± 5 g were housed under standard environmental conditions (23 ± 1 °C, 55 ± 5% humidity and a 12 h light/dark cycle) and maintained with free access to water and a standard diet ad libitum. The permission for animal utilization was obtained by the ethical committee of Oran University. The general guidelines for the care and use of laboratory animals recommended by the Council of European Communities (1987) were followed. Eighteen rats were divided into two groups. An experimental group (n = 12) was fed a diet containing 20% casein (C) combined with 5% of a mixture of vegetable oils (78% olive + 20% nut + 2% sunflower) containing 13% saturated fatty acids (SFA), 58% of monounsaturated fatty acids (MUFA) and 29% of polyunsaturated fatty acids (PUFA) with a ratio n-6/n-3 = 7 and supplemented with 1.5% of dietary cholesterol and 0.75% cholic acid to facilitate cholesterol absorption, for 10 days. After this period, hypercholesterolemic rats (total cholesterol (TC) value = 4.86 ± 1.19 mmol l1, were then randomly divided into two groups (n = 6), and fed for 4 weeks diets containing (g/100 g diet) 20% highly purified sardine proteins (SPc) or casein (CASc), with 5% mixture of vegetable oils, 1.5% cholesterol and 0.75% cholic acid. A control group (n = 6) was fed a standard diet containing 20% casein without cholesterol (CAS). Body weight and food consumption were recorded weekly and daily, respectively. The detailed composition of the diets and the characterization of the sardine protein extract (amino acids expressed in g/kg of protein) are shown in Table 1.

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Table 1 – Composition of purified diets (g/kg diet).a Components

SPc

CASc

CAS

b

– 200 577.5 40 50 50 40 20 15 7.5

200 – 577.5 40 50 50 40 20 15 7.5

200 – 600 40 50 50 40 20 – –

Casein Sardine proteinc Corn starchd Sucrosee Vegetable oilsf Celluloseg Mineral mixh Vitamin mixi Cholesterolj Cholic acidj a

The diets were isoenergetic (17.78 MJ/kg) and given in powdered form. Prolabo, Fontenay sous bois, France: provided the following amino acids as g/kg of protein: Arg: 36; His: 34; Ile: 42; Leu: 92; Lys: 74; Val: 57; Thr: 46; Phe: 49 Tyr: 53; Met:23; Cys: 2; Ala: 30; Asp: 66; Glu: 207; Gly: 18; Pro: 116; Ser: 55; Lysine/Arginine: 23; Methionine/Glycine: 1.5. c Proteins purified from sardine in our laboratory. Sardine protein provided the following amino acids as g/kg of protein (Garcia-Arias et al., 2003): Asp: 93; Thr: 47;Ser: 47; Glu: 120; Pro: 95; Gly:85; Ala:83; Cys:9; Val:53; Met:26; Ile:41; Leu:76; Tyr:24; Phe:31; His:40; Lys:75; Arg:45; Lysine/Arginine: 18; Methionine/Glycine: 3. d ONAB, Sidi Bel Abbe`s, Algeria. e Ce´vital, SPA, Bejaia, Algeria. f Market product: olive oil (15% SFA, 75% MUFA, 10% PUFA: 9% n-6 and 1% n-3) Ifri, Bejaı¨a, Algeria. Nuts oil (11% SFA, 18% MUFA, 71% PUFA: 58% n6 and 13% n-3) Lesieur, France. Sunflower oil (13% SFA, 22% MUFA, 65% PUFA: 65% n-6) Ce´vital, SPA, Bejaı¨a, Algeria. g Prolabo-Fontenay sous bois, France. h UAR 205 B (Villemoisson, 91360, Epinay/S/orge, France), mineral mix (mg/kg diet) CaHPO4, 17,200; KCl, 4000; NaCl, 4000; MgO2, 420; MgSO4, 2000; Fe2O3, 120; FeSO4Æ7H2O, 200; MnSO4ÆH2SO4ÆH2O, 98; CuSO4Æ5H2O, 20; ZnSO4, 80; CuSO4, 80; CuSO4Æ7H2O; KI, 0.32. i UAR 200 (Villemoisson, 91360, Epinay/S/Orge, France), vitaminic mix (mg/kg diet): Vit A, 39,600 UI; Vit D3, 5000 UI; Vit B1, 40; Vit B2, 30; Vit B3, 140; Vit B6, 20; VitB7, 300; Vit B12, 0.1; Vit C, 1600; Vit E, 340; Vit K, 3.80; Vit PP, 200; choline, 2720; folique acid, 10; para-aminobenzoic acid (PAB), 180; Biotine, 0.6. j Merck, Darmstadt, Germany. b

To evaluate the digestibility of lipids in hypercholesterolemic diet, particularly cholesterol, six animals from each group were placed individually into metabolism cages. Feces were collected from day 21 to day 28 of experiment. Total lipids were extracted according to method of Folch, Lees, and Sloane Stanley (1957).

2.3.

Sample collection

After 4-weeks of the experiment and overnight fasting between 07.00 and 8.00 h, six rats from each group were anesthetized with sodium pentobarbital (60 mg/kg body weight). Blood was collected from the abdominal aorta into tubes and serum was prepared by low-speed centrifugation (1000g for 20 min, 4 °C). Liver was removed immediately, rinsed with cold saline, and weighed. A serum aliquot was preserved in tubes containing 0.1% Na2 EDTA and 0.02% sodium azide for lipoproteins assays and another aliquot was used to measure LCAT activity. Liver samples were stored at 70 °C until use in the week.

2.4.

Lipids analysis

Serum, liver and fecal total lipids were determined by Folch method (1957). Total cholesterol (TC) and triacylglycerols (TG) were estimated by enzymatic colorimetric methods (kit CHOD-PAP Biocon, Vo¨nl-Marienhagen, Germany). Unesterified cholesterol (UC) was estimated by enzymatic method (kit CHOD-PAP Wako Chemicals, Richmond, VA, USA). Esterified cholesterol (CE) levels were calculated from the difference between TC and UC. Cholesteryl esters (EC) amounts were

estimated as 1.67 times the CE amounts. Analysis of phospholipids (PL) was assessed by enzymatic determination of PLs (kit cypress, Langdorp, Belgium).

2.5.

Isolation and characterization of serum lipoproteins

The lipoprotein fractions were separated by the precipitation method. Serum VLDL and LDL–HDL1 were isolated by precipitation using MgCl2 and phosphotungstate (Sigma Chemical Company, Lyon, France) by the method of Burstein, Scholnick, and Morfin (1970). HDL2 and HDL3 were separated by precipitation according to the method of Burstein, Fine, Atger, Wirbel, and Girard-Globa (1989) using MgCl2 and dextran sulfate MW 500,000 Da (Sigma Chemical Company). TC of each fraction and UC, PL and CE of HDL subfractions were determined by enzymatic colorimetric method described previously (kit CHOD-PAP Biocon, Vo¨nl-Marienhagen, Germany, kit CHOD-PAP Wako Chemicals, Richmond, VA, USA, kit cypress, Langdorp, Belgium). Protein contents of HDL3 were estimated according to the method of Lowry, Rosebrough, Farr, and Randall (1951) using bovine serum albumin as a standard.

2.6.

Assay of lipid peroxidation in serum and lipoproteins

Lipid peroxidation was estimated by measuring thiobarbituric acid reactive substance (TBARS) concentrations (Quintanilha, Packer, Szyszlo, Racanelly, & Davies, 1982) using tetraethoxypropane (Prolabo, Fontenay sous bois, France) as a precursor of malondialdehyde. One milliliter of diluted serum (protein concentration near 2 mg/ml) was

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added to 2 ml of thiobarbituric acid (final concentration in the mixture, 0.017 mmol/l (thiobarbituric acid 0.375 g was diluted in a solvent mixture composed by trichloroacetic acid 15%, chloridric acid 0.25 N and distilled water to a final volume of 100 ml) and butylated hydroxytoluene (final concentration, 3.36 lmol/l) and incubated for 15 min at 100 °C. After cooling samples were centrifuged, the absorbance of supernatant was measured at 532 nm.

2.7.

Assay for LCAT activity

LCAT activity was determined on fresh serum by an endogenous method (Albers, Chen, & Lacko, 1986). This procedure was based on the disappearance of the molecules of UC which were transformed into EC after the action of the LCAT. UC was evaluated by colorimetric enzymatic method (kit CHOD-PAP Wako Chemicals, Richmond, VA, USA).

3.3.

Serum and lipoprotein lipid peroxidation

Statistical analysis

Values were given as means ± S.E.M. of six rats per group. Statistical evaluation of the data was carried out by STATISTICA (Version 5, 1, Statsoft, Tulsa, OK, USA). After analysis of variance (ANOVA) the classification of the means was performed using Duncan’s new multiple range test (1955). The means with different superscript were considered significantly different (P < 0.05).

3.

Results

3.1.

Body weight, food intake and liver weight

The study of lipid peroxidation showed that serum TBARS concentrations were 1.7-fold higher with highly purified sardine protein (SPc) compared to casein with (CASc) or without (CAS) cholesterol (Table 5). In VLDL fraction TBARS contents were similar in the three groups, whereas their values were, respectively, 3.5- and 1.7-fold higher in LDL–HDL1 in rats fed SPc diet compared to CASc and CAS, (Table 5). However, in HDL2 and HDL3 TBARS levels were, respectively, 2.3- and 1.6fold lower in SPc compared which CASc.

3.5.

After 28 days of the experiment, highly purified sardine protein (SPc), casein (CASc) and control (CAS) group exhibited similar body weight and food consumption (Table 2). However, liver weights were increased by +28% and +14%, in SPc and CASc compared to CAS group, respectively (Table 2).

3.2.

Serum and lipoprotein lipid concentrations

At day 28, serum and liver TC and TG concentrations were not affected by the different diets (Table 4). In contrast, when compared with the baseline values corresponding to the beginning of the experiment (TC = 4.86 ± 1.19 and TG = 1.57 ± 0.07 mmol l1), serum TC and TG content were, respectively, 2- and 2.3-fold lower in SPC and CASc groups. Highly purified sardine proteins or casein diet supplemented with cholesterol induced a significant increase in LDL–HDL1C (+43%) as compared to the control group. However, VLDLC was similar in the three groups. HDL2-C concentrations were not significantly different with both dietary proteins supplemented with cholesterol. In addition, highly purified sardine proteins increased significantly HDL2-C (+42%) contents when compared with the CASc group.

3.4. 2.8.

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Lipid digestibility

At the end of the experiment, lipids intake values were increased by 35% and 18%, respectively, in the SPc and CASc groups compared with CAS. However, the digestibility was similar in the three groups (Table 3). The foecal cholesterol was, respectively, 5.3- and 8.4-fold higher in rats fed SPc and CASc diets compared to the control group. Moreover, the value noted in SPc was 1.6-fold lower than CASc group.

LCAT activity, HDL3-UC, -PL, -apo and HDL2-CE

In SPc group LCAT activity was, respectively, 1.5- and 1.6-fold higher compared to CASc and CAS values (Table 6). Compared with the SPc group, HDL3-PL contents were 1.4-fold increased in CASc and CAS. Moreover, HDL3-UC contents of the rats fed CASc and CAS diets were, respectively, 1.8- and 1.5-fold higher compared to SPc values. Furthermore, the HDL2-CE concentrations were, respectively, 1.8- and 1.4-fold higher in SPc group compared with CASc and CAS.

4.

Discussion

In the present study, we examined whether highly purified sardine proteins might improve reverse cholesterol transport and reduce lipoproteins oxidative damage induced by a highcholesterol diet. To approach the Mediterranean diet model, we selected in our experiment a mixture of vegetable oils

Table 2 – Body and liver weights and food intake in rat fed experimental diets for 28 days. Diets

Body weight (g) Food intake (g/day/rat) Liver weight (g)

SPc

CASc

CAS

173 ± 22 12.80 ± 2.02 9.11 ± 0.64a

160 ± 7 12.00 ± 1.73 8.27 ± 0.81a

154 ± 4 12.00 ± 1.15 7.06 ± 0.36b

Data are shown as the mean ± S.E.M. for six values per group. Two groups (n = 6) fed a cholesterol-enriched diet containing 20% sardine proteins (SPc) or casein (CASc). A control group (CAS) (n = 6) was fed a 20% casein diet. Statistical analysis was performed using the Duncan’s multiple range test. The means with different superscript were considered significantly different (P < 0.05).

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Table 3 – Dietary and fecal contents of lipids and cholesterol in rat fed experimental diets for 28 days. Diets SPc

CASc a

Lipids intake (mg/day/rat) Fecal lipids (mg/day/rat) Cholesterol intake (mg/day/rat) Fecal cholesterol (mg/day/rat) Lipid digestibility (%)

CAS a

815 ± 49.50 80.78 ± 17.62a 190 ± 8.66 18.46 ± 0.11b 90.09 ± 2.77

605 ± 106.07b 24.40 ± 0.84b – 3.48 ± 0.74c 92.43 ± 1.64

715 ± 183.85 67.65 ± 3.96a 165 ± 42.43 29.36 ± 3.73a 90.29 ± 1.94

Data are shown as the mean ± S.E.M. for six values per group. Two groups (n = 6) fed a cholesterol-enriched diet containing 20% sardine proteins (PSc) or casein (CASc). A control group (CAS) (n = 6) was fed a 20% casein diet. Statistical analysis was performed using the Duncan’s multiple range test. The means with different superscript were considered significantly different (P < 0.05). lipidsexcreted lipids  100. Lipids digestibility ¼ ingestedingested lipids

Table 4 – Serum and liver lipids and lipoprotein cholesterol in rat fed experimental diets for 28 days. Diets SPc

CASc

CAS

1

Serum lipids (mmol l ) Total cholesterol Triacylglycerols VLDL-C LDL-C HDL2-C HDL3-C

2.68 ± 0.50 0.67 ± 0.24 0.59 ± 0.05 1.12 ± 0.26a 0.55 ± 0.05a 0.36 ± 0.01a

2.37 ± 0.42 0.67 ± 0.16 0.58 ± 0.03 1.19 ± 0.03a 0.32 ± 0.03b 0.32 ± 0.01a,b

1.86 ± 0.26 0.84 ± 0.13 0.57 ± 0.02 0.66 ± 0.01b 0.41 ± 0.03a,b 0.30 ± 0.02b

Liver lipids (lmol g1) Total cholesterol Triacylglycerols

133.13 ± 26.80 104.94 ± 32.52

102.63 ± 30.38 96.20 ± 31.13

99.92 ± 13.08 73.90 ± 34.89

Data are shown as the mean ± S.E.M. for six values per group. Two groups (n = 6) fed a cholesterol-enriched diet containing 20% sardine proteins (SPc) or casein (CASc). A control group (CAS) (n = 6) was fed a 20% casein diet. Statistical analysis was performed using the Duncan’s multiple range test. The means with different superscript were considered significantly different (P < 0.05).

Table 5 – Thiobarbituric acid reactive substances (TBARs) contents in serum and lipoproteins (lmol ml1) in rat fed experimental diets for 28 days. Parameter

SPc

CASc a

Serum VLDL LDL–HDL1 HDL2 HDL3

CAS b

26.81 ± 2.85 91.81 ± 4.93 117.62 ± 7.89a 49.25 ± 21.70b 211.81 ± 6.90b

17.15 ± 1.69b 149.02 ± 60.18 70.2 ± 14.84b 32.7 ± 2.12b 99.48 ± 1.97c

15.80 ± 3.59 139.25 ± 50.31 33.45 ± 7.42c 114.83 ± 25.65a 348.55 ± 20.71a

Data are shown as the mean ± S.E.M. for six values per group. Two groups (n = 6) fed a cholesterol-enriched diet containing 20% sardine proteins (SPc) or casein (CASc). A control group (CAS) (n = 6) was fed a 20% casein diet. Statistical analysis was performed using the Duncan’s multiple range test. The means with different superscript were considered significantly different (P < 0.05).

Table 6 – LCAT activity, HDL3-UC, HDL3-PL, HDL3-apo and HDL2-CE in rat fed experimental diets for 28 days. Parameter

SPc 1

LCAT activity (nmol HDL3-UC (mmol l1) HDL3-PL (mmol l1) HDL3-apo (g l1) HDL2-CE (mmol l1)

h

1

ml

1

serum)

CASc a

69.60 ± 14.49 0.038 ± 0.006b 0.669 ± 0.003b 0.270 ± 0.007a 0.87 ± 0.09a

CAS b

45.82 ± 5.14 0.069 ± 0.007a 0.906 ± 0.129a 0.222 ± 0.002c 0.48 ± 0.04c

42.92 ± 7.70b 0.058 ± 0.003a 0.927 ± 0.003a 0.244 ± 0.004b 0.61 ± 0.05b

Data are shown as the mean ± S.E.M. for six values per group. Two groups (n = 6) fed a cholesterol-enriched diet containing 20% sardine proteins (SPc) or casein (CASc). A control group (CAS) (n = 6) was fed a 20% casein diet. Statistical analysis was performed using the Duncan’s multiple range test. The means with different superscript were considered significantly different (P < 0.05).

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(78% olive + 20% nut + 2% sunflower) to approach foodstuffs characterizing the Mediterranean diet that is recognized for its beneficial effects on cholesterol metabolism and lipoprotein oxidation (Razquin et al., 2009). In the present investigation, sardine protein compared to casein involved a similar body weight in spite of the same food intake (Table 2). In agreement with previous studies (Bastida, Garcı´a-Linares, Viejo, Garcı´a-Arias, & Sa´nchezMuniz, 2006), data suggest that sardine protein permits a normal body increase and well accepted by the rats. The fact that lipids digestibility was similar in rats fed sardine protein compared with those fed casein indicated that lipids utilization was not altered by the dietary cholesterol or by the origin of proteins consumed by the studied groups. At the end of the experiment, highly purified sardine proteins exerted a similar cholesterolemia compared with casein diets. In SPc and CASc, the higher serum TC concentration noted at the beginning of this study decreased after 28 days of the experiment and reached the control values. This could be explained by an adaptation to these diets by rats and to the variety of oil combined with sardine or casein protein in the diet. This result might suggest a decrease in cholesterol absorption and/or an increase in fecal steroid excretion. These results are in agreement with several studies in animals that consumed cholesterol enriched diet which reported that the mechanisms that prevent dyslipidemia in rats are due to the reduction of the feedback inhibition of cholesterol biosynthesis and increased bile acid excretion, leading to a minor elevation of serum cholesterol (Kritchevsky, 1995). Numerous data from both epidemiological and experimental origins indicate that some dietary proteins and amino acids in supplements can modify blood LDL cholesterol, HDL cholesterol and total cholesterol (Blachier, Lancha, Boutry, & Tome, 2010). Indeed, two essential amino acids, lysine and methionine, were most effective for increasing serum cholesterol. Studies dealing with the effects of fish protein on lipid metabolism suggest that specific amino acids could be responsible for several effects observed in rabbits (Kritchevsky et al., 1982) and in rats (Lavigne et al., 2000). Kritchevsky et al. (1982) suggested that the lower lysine/ arginine ratio of fish protein compared to casein could be responsible for a hypocholesterolemic effect in rabbit. Some studies carried out in rats showed well the impact of this ratio in cod protein (Lavigne et al., 2000) and whiting protein (Shukla et al., 2006). In spite, high content of glycine, arginine and the lower methionine/glycine ratio in sardine protein compared with casein, our results do not show a change in cholesterolemia. Other possible mechanisms could be explained: an increase in conversion of cholesterol into bile acids by cholesterol 7 a-hydroxylase stimulation and/or an inhibition or a reduction of cholesterol endogenous synthesis by hydroxy-methyl-glutaryl-CoA (HMG-CoA) reductase activity and/or an increase in acyl-CoA cholesterol acyltransferase (ACAT) activity (enzyme involved in the esterification of cellular cholesterol). Sardine proteins are rich in taurine (Shirai, Terayama, & Takeda, 2002) compared with cod, milk casein, soy and whey proteins (Wo´jcik, Koenig, Zeleniuch-Jacquotte, Costa, & Chen, 2010) which may enhance the biotransformation of cholesterol to bile acids. Moreover, several studies have shown that

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taurine has a hypolipidemic effect (Park, Kyungshin, & Youngsook, 1998). This assumption could explain the attenuation of hypercholesterolemia in rats fed purified sardine proteins. These results showed that there were interactions between dietary proteins and lipids in the regulation of serum cholesterol levels in the rat. The fatty acids composition of plant oils mixture (olive–nut–sunflower) used in our study had probably masked the effect of dietary proteins on hypercholesterolemia. Indeed, from the various studies carried out in rat, it appears that fish protein has variable effects on cholesterolemia and lipoproteins profile according to the type, the quantity and nature of lipids contained in diets (Demonty et al., 2003; Zhang & Beynen, 1993). However, the cholesterol content of VLDL–LDL and HDL fractions remained unchanged. In our study, there was no effect of highly purified sardine proteins on LDL-cholesterol concentration; this is in contrast with the published results that showed a distinct reduction of plasma and/or LDL-cholesterol after administration of fish protein in men (Lavigne et al., 2000), and animals (Zhang & Beynen, 1993). The increase in HDL-cholesterol observed with SPc compared with CASc contradicts the findings in hypercholesterolemic rats fed sardine proteins with 2% of cholesterol and combined with 10% olive oil (Bastida et al., 2006) and even in rats fed whiting protein combined with 10% lard and supplemented with 0.5% cholesterol (Shukla et al., 2006). This discrepancy may be due to differences across the experimental conditions (e.g., rat strains used and the presence or absence of cholesterol in the diet). Serum TBARS concentrations, a marker of lipid peroxidation, were higher with highly purified sardine proteins compared to casein with or without cholesterol diet. This finding is attributed, in part to the hypercholesterolemic and hypertriacylglyceridemic effects induced by this high dietary cholesterol consumption. Highly purified sardine proteins reduced VLDL oxidation but not significantly, whereas they increased it in LDL–HDL1. Hence, the enhanced oxidation of LDL particles in rats fed highly purified sardine proteins compared with casein, may probably result from their high cholesterol and phospholipid (PL) contents (data not shown), which in turn could expose them to free radical attacks. Recent investigations suggest that HDL plays a key role in the protection of LDL from oxidation. Such activity depends on the presence of apolipoproteins (apoA-I, apoA-II, apoAIV, apoE) and enzymes (paraoxonase 1, platelet activating factor-acetyl hydrolase, LCAT, glutathione peroxidase) (Navab, Reddy, Van Lenten, Anantharamaiah, & Fogelman, 2009). In spite of the high levels of TBARS in LDL particles, it seems that HDL particles were not affected by lipid peroxidation. Indeed, the highly purified sardine proteins induced a significant decrease in TBARS content of HDL2 and HDL3. In the present study, LCAT activity was significantly higher in hypercholesterolemic rats fed highly purified sardine proteins compared to casein supplemented with cholesterol and to the control group. Moreover, serum HDL2-CE content (product of enzymatic reaction) was increased. However, a reduction in HDL3-PL (substrate of LCAT) and HDL3-UC (acceptor of lecithin acyl group) was noted. The enhanced LCAT activity could possibly be related to an increased expression of its apo AI cofactor activator and/or its hepatic genes synthesis in the liver. This assumption is supported by Shukla et al. (2006) who

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showed that the whiting protein compared to casein had a higher relative hepatic gene involved in the synthesis of apo AI and LCAT. Feeding purified sardine proteins enhance probably an efficient flow of free cholesterol from peripheral tissues to plasma via the HDL2 rich in cholesteryl esters. Indeed, the higher level of HDL2-CE could probably create a gradient necessary for this transfert, involving thus a high efficacy of reverse cholesterol transport. Some reports have mentioned that LCAT enzyme may have a dual role in lipoprotein oxidation, whereby it acts in a dose-responsive manner as a potent prooxidant during VLDL oxidation, but as an antioxidant during LDL oxidation (McPherson et al., 2007). In hypercholesterolemic rats fed highly purified sardine proteins compared with casein (Table 6), LDL oxidation was higher despite the enhanced LCAT activity. These findings suggest that highly purified sardine proteins do not play an important role in serum and LDL oxidation but it seems to prevent the antiatherogenic fraction from free radical attacks. In our experimental conditions, the surprising results that noted with sardine proteins, promoted oxidation in LDL, but retarded oxidation of HDL. These results which seem paradoxical could be explained by the fact that phospholipids content was lower in HDL and consequently became unable to reduce LDL oxidation. Ansell et al. (2005) have reported that phospholipids in the HDL3 fraction are especially capable of retarding LDL oxidation. Moreover, the low PL content noted in HDL was due probably to the higher LCAT activity. Nevertheless, at the same time this enhanced enzyme activity promoted an efficient cholesterol efflux by HDL-CE rich toward liver for excretion in bile acids form. This work is the first report of the effects of highly purified sardine (S. pilchardus) proteins on lipid peroxidation and reverse cholesterol transport in rats fed high-cholesterol diet. Highly purified sardine proteins have no beneficial effects on LDL-cholesterol and lipid peroxidation. However, they reduce HDL oxidation and improve reverse cholesterol transport, in the hypercholesterolemic rats.

Acknowledgment This research was supported by the Algerian Ministry of Higher Education and Scientific Research.

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