Effect of amidated alginate on faecal lipids, serum and hepatic cholesterol in rats fed diets supplemented with fat and cholesterol

Accepted Manuscript Effect of amidated alginate on faecal lipids, serum and hepatic cholesterol in rats fed diets supplemented with fat and cholesterol

Milan Marounek, Zdeněk Volek, Tomáš Taubner, Dagmar Dušková, Ladislav Čermák PII: DOI: Reference:

S0141-8130(18)33158-1 https://doi.org/10.1016/j.ijbiomac.2018.10.180 BIOMAC 10831

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

25 June 2018 21 October 2018 25 October 2018

Please cite this article as: Milan Marounek, Zdeněk Volek, Tomáš Taubner, Dagmar Dušková, Ladislav Čermák , Effect of amidated alginate on faecal lipids, serum and hepatic cholesterol in rats fed diets supplemented with fat and cholesterol. Biomac (2018), https://doi.org/10.1016/j.ijbiomac.2018.10.180

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ACCEPTED MANUSCRIPT Effect of amidated alginate on faecal lipids, serum and hepatic cholesterol in rats fed diets supplemented with fat and cholesterol Milan Marounek*, Zdeněk Volek, Tomáš Taubner, Dagmar Dušková, Ladislav Čermák Contact information: Department of Nutrition Physiology and Animal Product Quality, Institute of Animal Science, CZ-104 00, Praha-Uhříněves, Czech Republic

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*Corresponding author. Tel.: +420 776 053 360 E-

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mail address: [email protected]

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ABSTRACT

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The effect of octadecylamide of alginic acid on serum and hepatic cholesterol, and the faecal output of fat and sterols was examined in female rats fed diets containing cholesterol and palm fat at 10 and 50

g

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kg -1 , respectively. Cholesterol supplementation significantly increased serum and hepatic cholesterol concentrations, and faecal output of cholesterol and coprostanol. Cholesterol and amidated alginate

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supplementations changed the profile of fatty acids in the faeces. Molar percentages of saturated fatty acids,

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palmitic and stearic acid in rats fed diets supplemented with cholesterol were increased and those of unsaturated fatty acids were decreased. Cholesterol increased molar percentages of saturated fatty acids and

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amidated alginate reversed this effect. Amidated alginate, supplied at 10, 20 and 40 g kg-1, significantly decreased serum cholesterol from 2.82 to 2.00, 1.95, and 1.63 µmol mL-1, respectively, and significantly

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decreased hepatic cholesterol from 13.8 to 9.33, 7.81 and 6.3 µmol g-1, respectively. Amidated alginate

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increased the faecal output of fat and neutral sterols in a dose-dependent manner. In contrast, the output of bile acids was significantly decreased. The faecal outputs of fat and serum cholesterol were negatively correlated. At the highest concentration tested, amidated alginate significantly reduced the serum concentration of triacylglycerols. It can be concluded that amidated alginate is an effective cholesterollowering agent and sorbent of dietary fat.

Keywords: Amidated alginate · Faeces · Fatty acids · Liver · Rats · Serum lipids

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ACCEPTED MANUSCRIPT 1. Introduction Alginate is a linear copolymer composed of (14) linked β-mannuronic acid and -Lguluronic acid, present in the cell walls of brown algae [1]. Alginate has many uses in the food industry, as a thickening agent and a gelling agent in jams and jellies [2]. Alginate is classed as

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a dietary fibre which slows gastric clearance, moderate appetite, and attenuate uptake of

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nutrients from the small intestine [3]. Alginate thus could be used as a potential weight loss

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treatment when added to food. In addition, alginate seems to exhibit beneficial influence on

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postprandial glucose absorption and insulin response in animals and humans [4]. In the rat, alginate showed a tendency to decrease the hepatic cholesterol level and increase

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faecal cholesterol excretion [5]. In rats fed a high-cholesterol diet calcium alginate was effective in reducing plasma cholesterol [6]. Previous experiment showed that in rats fed a diet

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supplemented with cholesterol and palm fat, alginate from brown algae at 20 g kg-1 reduced

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hepatic cholesterol, but did not influence serum cholesterol, serum triacylglycerols and total

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hepatic lipids. Changing hydrophilic alginate to its hydrophobic derivate (n-octadecylamide of alginic acid) modified alginate significantly decreased serum cholesterol, serum triacylglycerols

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and total hepatic lipids. Amidated alginate significantly increased the faecal concentration of neutral sterols and showed a tendency to increase the faecal concentration of fat [7].

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The aim of the present study was to assess the dose-dependent effect of amidated alginate on serum lipids, hepatic cholesterol and faecal output of lipids. A hypothesis was tested that serum cholesterol in rats fed cholesterol and fat depends on the amount of fat leaving the body in faeces. 2. Materials Sodium alginate from brown algae was purchased from Sigma-Aldrich (a low-viscosity 2

ACCEPTED MANUSCRIPT product A2158, Prague, Czech Republic). Alginic acid was prepared by washing sodium alginate with 1:1 mixture of ethanol and 4 M HCl, pure ethanol, aceton, and dried in air. Alginic acid was esterified with methanol containing concentrated sulphuric acid (1mL in 200 mL). The reaction mixture was stirred at 60 °C for 72 h, filtered, and the product was washed with ethanol,

n-octadecylamine and

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octadecylamine [8]. Briefly, amidation reagent was prepared from

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acetone and dried in air. The methyl ester of alginic acid was amino-dealkoxylated with n-

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dimethylformamide by stirring at 55 °C for 30 min, then methyl ester of alginic acid was added and the reaction was carried out at the same temperature for 72 h. The product of the reaction was

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filtered, and thoroughly washed with petroleum ether and pure ethanol. Its degree of amidation,

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calculated according to Taubner et al. [9] on basis of organic elemental analysis was 94.3%. The rat diet ST-1 was supplied by Velaz Ltd. (Lysolaje, Czech Republic). Protected palm fat

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AkoFeed Gigant 60 was obtained from AarhusKarlshamn Sweden AB (Prague, Czech Republic).

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Sylon HTP kit (hexamethyldisilazane-trimethylchlorsilan-pyridine 3:1:9) was purchased from Supelco (Bellefonte, U.S.A.). Isolithocholic acid, 12-ketolithocholic acid, norcholic acid, α-, β-

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, ω-muricholic acids, and β-sitostanol were purchased from Steraloids Inc. (Newport, U.S.A.).

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Other sterols were supplied by Sigma-Aldrich (Prague, Czech Republic).

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2.1. Animals and diets

Thirty female Wistar rats, approximately 6 weeks old, were used. The rats were housed individually in a temperature- and humidity-controlled room. The vivarium was maintained on a 12 h light: 12 h dark photoperiod cycle at a temperature of 22 ± 1 °C. The ST - 1 rat diet was supplemented with microcrystalline cellulose and palm fat at 60 and 40 g kg-1, respectively (basal diet, no. 1). After 4 weeks, the rats were randomly divided into 5 groups of 6 rats each. The diets no. 2, 3, 4, and 5 were supplemented with cholesterol at 10 g kg -1 , at the expense of 3

ACCEPTED MANUSCRIPT palm fat. The diets no. 3, 4, and 5 were supplemented with amidated alginate at 10, 20, and 40 g kg-1, respectively, at the expense of cellulose (Table 1). Two diets were control diets (a diet without supplements and a diet with cholesterol but without amidated alignate).

Table 1

Diets and water were available ad libitum. The experiment duration (3 weeks) and dosing of

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cholesterol (0 or 10 g kg-1) did not differ from our previous experiment [7]. Then the rats were

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sacrificed by decapitation, after anaesthesia by the inhalation of Isofluran (Nicholas Piramal

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India Ltd., London, U.K.). The rats received 4 g of feed 4 h before they were sacrificed. The body weight and feed intake were measured. The study was approved by the Ethics

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Committee of the Institute of Animal Science.

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2.2. Sampling

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Mixed blood samples were collected from each rat at the time of slaughter. The samples were taken into tubes without anticoagulant and allowed to stand for 30 min. The sera were

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separated by centrifugation, stored at +4 °C, and analysed the subsequent day. The livers were

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excised and kept at –40 °C until analysis. Faeces were collected during the last 5 days of the

2.3. Analyses

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experiment, weighed, pooled and stored frozen until analysis.

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The serum cholesterol, triacylglycerols, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined using commercial kits (BioVendor Ltd., Brno, Czech Republic). The total hepatic and faecal lipids were extracted with 2:1 chloroform-methanol and measured gravimetrically [10]. Hepatic cholesterol was saponified with 2M ethanolic KOH (1 h at 100 °C), extracted with ethyl ether, derivatised using trimethylchlorosilane

and

hexamethyldisilazane (Sigma-Aldrich, Prague, Czech Republic), and determined using gas chromatograph GC 7890 A-system (Agilent Technologies, Inc., Santa Clara, U.S.A.), equipped 4

ACCEPTED MANUSCRIPT with SAC-5 capillary column (Supelco, Bellefonte, U.S.A.), operated isothermally at 285 °C. The 5α-cholestane at 0.5 mg mL-1 was used as the internal standard. Faecal samples of rats fed diets no. 1,2,4,5 were freeze-dried and used for the fatty acids (FA) determination, after alkaline trans-methylation according to ISO 5509 [11]. The HP 6890N gas

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chromatograph equipped with a programmed (150-230 °C) 60 m Db-23 capillary column

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(Agilent Technologies, Inc., Santa Clara, U.S.A.) was employed. Nonadecanoic acid (C 19:0)

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was used as an internal standard to quantify FA present in samples. FA were identified by retention times compared with standards PUFA 1, PUFA 2, PUFA 3 and 37 component FAME

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Mix (Supelco).

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Faecal sterols were determined in freeze-dried samples chromatographically after butylation and silylation according to Batta et al. [12]. Norcholic acid was used as the internal standard. N-

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butanol (4 mL) and norcholic acid (1mg) were added to 350 mg samples, followed by the addition

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of concentrated HCl (0.8 mL). The mixture was heated in sealed vessels for 4h at 60 °C, centrifuged, and the supernatant (220 µL) was evaporated at 60 °C under N2. The residue was

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subjected to trimethylsilylation with 200 µL of Sylon HTP at 55 °C for 30 min, followed by

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evaporation at 60 °C under N2. The residue was taken up in 2 mL of hexane, centrifuged and 12 µL of supernatant was injected into the GC. Thermo Scientific gas chromatograph Focus GC

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coupled to a Thermo ITQ mass-selective detector was used. The chromatograph was equipped with the Thermo TR-5MS SQC column (30 m x 0.25 mm i.d.). Helium at a flow rate of 1.0 mL min-1 was the carrier gas. After injection, oven temperature was kept at 120 °C for 4 min, then programmed at 6 °C min-1 to a final temperature of 272 °C, and held for 45 min. The neutral sterols and bile acids were identified on the basis of retention times by comparing to standards. The linear detector responses were confirmed for variable amounts of individual neutral sterols and bile acids injected onto the column. 5

ACCEPTED MANUSCRIPT 2.4. Statistics The data were analysed by a one-way analysis of variance using the GLM procedure of SAS, version 8.2 (SAS Institute, Cary, NC, U.S.A.). The results were expressed as the mean and the SEM. Significant differences (P < 0.05) were identified using Tukey´s test. The Pearson

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correlation coefficient was used as a measure of the dependence between pairs of observations.

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3. Results

Body weight and feed intake did not differ among treatment groups in this experiment

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(Table 2). Supplementation of diets with cholesterol significantly increased total and LDL

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cholesterol, as well as cholesterol concentration in the liver tissue. In rats fed diets supplemented with cholesterol, amidated alginate significantly decreased serum total cholesterol and hepatic

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cholesterol. The serum total cholesterol and hepatic cholesterol were significantly correlated (r

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= 0.623; P < 0.001). At the highest concentration tested (40 g kg-1), amidated alginate significantly reduced serum concentration of triacylglycerols. There was no significant effect

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of amidated alginate on activity of aminotransferases and weight of liver.

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Table 2

In rats fed cholesterol, amidated alginate significantly increased faecal daily output of fat,

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coprostanol, and neutral sterols in a dose-dependent manner (Table 3). Daily faecal fat output represented 20.0 - 44.6% of the fat intake. The faecal output of fat and serum cholesterol were Table 3

negatively correlated (r = -0.402; P = 0.027).

Fig. 1 presents the relationship of serum cholesterol and daily output of fat. The faecal output of cholesterol and serum cholesterol were correlated non-significantly (r = -0.117; P = 0.538). This correlation, however, was significant in rats fed five diets supplemented with cholesterol (r = -0.532; P = 0.008).

Fig 1 6

ACCEPTED MANUSCRIPT The faecal daily output of bile acids was significantly decreased in rats fed diets supplemented with amidated alginate. The faecal output of bile acids significantly correlated with the concentration of hepatic cholesterol (r = 0.575; P < 0.001), but non-significantly with the fat intake (r = 0.326; P = 0.079).

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Cholesterol supplementation significantly increased molar percentages of palmitic acid, stearic

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acid, and saturated FA in faeces. Molar percentages of oleic acid (C 18:1 n9), linoleic acid and

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unsaturated FA (monounsaturated FA and polyunsaturated FA) were significantly decreased in faeces of rats fed cholesterol-containing diets. Amidated alginate at 40 g kg-1 significantly

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decreased molar proportion of palmitic acid and SFA in faecal FA, whereas proportion of MUFA was increased (Table 4).

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Table 4

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4. Discussion

The hypocholesterolaemic effect of amidated alginate in rats fed cholesterol- and fat-

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enriched diets was similar to that observed in previous experiments using diets supplemented

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with amidated pectin [13, 14], and amidated celluloses [15]. Modified polysaccharides also significantly decreased cholesterol concentration in the liver tissue, and at the highest dosing

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(i.e., amidated alginate at 40 g kg-1, amidated pectin and amidated carboxymethylcellulose at

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60 g kg-1), also decreased the serum concentration of triacylglycerols. Amidated alginate was a better sorbent of fat than amidated pectin. Amidated pectin at 60 g kg-1 increased faecal concentration of fat by 51% [16], whereas a comparable effect was observed in rats of the same age fed amidated alginate at 20 g kg-1 (present study). The negative correlation between the faecal output of fat and serum cholesterol is a new finding, which is consistent with the opinions of Lichtenstein et al. [17] and Dietschy [18], who concluded that the reduction of total fat and saturated fatty acid intake was pivotal for the reduction of serum total and LDL cholesterol 7

ACCEPTED MANUSCRIPT concentrations. In rats fed diets containing cholesterol serum cholesterol negatively correlated with the faecal output of cholesterol. This indicates that the sequestration of cholesterol (which decreased its enterohepatic circulation) contributed to the hypocholesterolaemic effect of amidated alginate. Cholesterol is present in the fat leaving the body in faeces, thus, it is difficult

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to distinguish the effect of cholesterol sequestration and effect of losses of other lipids present

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in faeces. The faecal concentration of bile acids significantly decreased in treated rats, which

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suggests a lower synthesis in the liver in response to the lower availability of substrate cholesterol. Bile acid synthesis in rat hepatocytes might be regulated by the availability of

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cholesterol to cholesterol 7α-hydroxylase [19]. Hydroxylation of cholesterol to 7α-

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hydroxycholesterol is the rate-limiting step in synthesis of bile acids. Indeed, the faecal output of bile acids and cholesterol concentration in hepatocytes significantly correlated.

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The source of palmitate and stearate in faeces of rats fed diets rich in cholesterol are

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probably cholesterol esters leaving enterocytes in chylomicrons. Cholesterol esters are accumulated in HDL lipoproteins which are the most relevant source of cholesterol secreted

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into the bile [20]. The source of FA in faeces may be also the undigested fat.

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In the caecum of rats fed alginate Firmicutes was the most abundant phyllum and Abaculum was dominant genus [21]. Hydrophilic alginate changing into hydrophobic derivative

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(octadecylamide) greatly increased affinity to lipids; however, the solubility in water was lost (7). In the available literature there is a paucity of information on the effect of amidated polysaccharides on intestinal microbiota. In cultures of the pig colon content supplied with amidated derivatives of pectin the production of short-chain fatty acids (SCFA) and gas were lower than in control cultures of citrus pectin. Production of SCFA and gas correlated negatively with the degree of amidation. The length of the carbon chain of alkyls (C4 to C18) had no effect

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ACCEPTED MANUSCRIPT on fermentability of modified pectins. Amidation of pectin reduced the number of colon bacteria capable of growing on the pectin by one number of magnitude. Gramnegative rods were the most numerous pectinolytic isolates [22]. The amidation of alginate probably also limits the availability of modified alginate to the non-amidated part of the molecule (several %, in the present

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experiment). Bacteria utilizing alginate and non-amidated parts of the amidated alginate molecule

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are probably the same. The mainstay therapeutic interventions for hypercholesterolaemia are

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statins, drugs that inhibit the synthesis of cholesterol [23]. Lipid-lowering drugs of different classes have a synergistic effect on lipid metabolism, and combination therapy is often used

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[24]. The combination of statins with cholestyramine (a bile acids sequestrant) has been reported

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in several clinical studies [25-27]. Statins do not reduce cholesterol absorption; thus, adjunct therapy is feasible. There was no negative effect of amidated alginate on the feed intake,

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weight of liver, or activity of aminotransferases; however, the possibility of depletion of fat-

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soluble vitamins from the intestinal lumen at high dosing of amidated alginate cannot be

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excluded. 5. Conclusion

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In rats fed diets containing cholesterol and palm fat amidated alginate decreased serum and

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hepatic cholesterol, and increased faecal output of fat and neutral sterols. A significant part of the dietary fat left the body in faeces. The cholesterolaemia negatively correlated with the faecal output of fat, and in cholesterol-fed rats also with the faecal output of cholesterol. Taken together, amidated alginate is an effective cholesterol-lowering agent and sorbent of dietary lipids. Acknowledgements This work was supported by the Ministry of Agriculture of the Czech Republic (Project MZERO0718). 9

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[2]

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ACCEPTED MANUSCRIPT (2010) 299-303. [14] M. Marounek, Z. Volek, D. Dušková, J. Tůma, T. Taubner, Dose-response efficacy and long-term effect of the hypocholesterolemic effect of octadecylpectinamide in rats, Carbohyd. Polym. 97 (2013) 772-775. [15] J. Tůma, Z. Volek, A. Synytsya, D. Dušková, M. Marounek, Hydrophobically modified celluloses as novel cholesterol-lowering polymers, BioResources 9 (2014) 4266-4273.

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ACCEPTED MANUSCRIPT with hypercholesterolemia, Arch. Inter. Med. 153 (1993) 1321-1329. [26] D.L. Sprecher, J. Abrams, J.W. Allen, W.F. Keane, S.G. Chrysant, H. Ginsberg, J.J. Fischer, B.F. Johnson, P. Theroux, L. Jokubaitis, Low-dose combined therapy with fluvastatin and cholestyramine in hyperlipidemic patients, Ann. Intern. Med. 120 (1994) 537-543. [27] M. Eriksson, K. Hadell, I. Holme, G. Walldius, T. Kjellstrom, Compliance with and

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AN

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ACCEPTED MANUSCRIPT Fig. 1. The relationship of serum cholesterol and faecal output of fat Table 1 Composition of control and experimental diets (g kg-1) Diet

a

a

2 10

3

4

5

10

10

10

Palm fat

60

50

50

50

50

Amidated alginate

0

0

10

20

40

Cellulose

40

40

30

20

900

900

900

900

Control diets without amidated alginate

900

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a

0

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Diet ST-1

b

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Cholesterol

1 0

b

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The rat diet ST-1 ingredients were soybean meal, meat and bone meal, fish meal, wheat, maize, oats, wheat bran, limestone, dicalcium phosphate, salt, and supplements of vitamins, trace elements and amino acids. The diet contained crude protein, fibre, fat, ash and cholesterol at 196, 40, 24, 41, and 0.28 g kg-1, respectively.

2 10

3 10

4 10

5 10

Amidated alginate (g kg-1)

0

0

10

20

40

Initial weight (g)

246

246

244

245

244

2.33

262

265

255

261

255

2.22

19.3

19.8

19.5

19.6

19.0

0.10

b

1.95b

1.63

b

b

Final weight (g) -1

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Feed intake (g day ) Serum concentrations

Total cholesterol (µmol mL-1) -1

2.04 0.30

Triacylglycerols (µmol mL-1)

0.97

-1

3.07

-1

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ALT (nkat mL )

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LDL cholesterol (µmol mL )

AST (nkat mL )

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1 0

ED

Diet Cholesterol (g kg-1)

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Table 2 Effect of cholesterol and amidated alginate on growth, feed intake, serum and hepatic lipids, aminotransferases, and liver weight in ratsa

-1

b b bc

2.82 0.76 1.30

c

2.00

c

0.24

bc

3.39

1.09

bc

2.74

1.08 b

1.34

1.43

b

3.27

1.03 c

0.23

0.70

b

0.10

b

0.05

c

0.08

3.68

1.32 d

0.33

SEM

0.12

1.18 de

0.04 e

Hepatic cholesterol (µmol g )

5.42

Hepatic fat (mg g-1)

50.5

70.3

60.6

50.2

61.9

2.57

Weight of liver (g per 100 g)

3.28

3.28

3.68

3.57

3.76

0.07

a Six

13.84

9.33

female rats per group in the same row with different superscripts differ significantly

b-eValues

13

7.81

6.30

0.74

ACCEPTED MANUSCRIPT Table 3 Effect of cholesterol and amidated alginate on daily faecal output of dry matter, fat, and sterols in rats a. 3 10 10 2.89 394cd 25.0d 134.0d 32.8d 185.6d 21.1e 206.7d

4 10 20 2.81 507d 32.0de 132.9d 46.6de 196.7de 21.7e 218.4d

a

5 10 40 2.92 685e 44.6e 149.4d 64.4e 229.7e 25.4e 255.1d

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2 10 0 3.09 319c 20.0c 126.8d 31.8d 175.7d 34.2d 209.9d

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1 0 0 2.69 332c 21.2c 7.8c 6.4c 27.1c 11.5c 38.6c

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Diet Cholesterol (g kg-1) Amidated alginate (g kg-1) Dry matter (g) Fat (mg) Fat (% of intake) Cholesterol (µmol) Coprostanol (µmol) Neutral sterolsb (µmol) Bile acids (µmol) Total sterols (µmol)

SEM 0.08 29.9 1.95 10.2 5.1 14.0 1.75 15.2

Six female rats per group. Including plant sterols c-e Values in the same row with different superscripts differ significantly.

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b

-1

Cholesterol (g kg ) Amidated alginate (g kg-1)

3.85

Fatty acid profile (mol.%)

0

a

PT 30.36

Palmitic (C 16:0)

AC

CE

Palmitoleic (C 16:1)

Linoleic (C 18:2)

10

1.20a

Myristic (C 14:0)

Stearic (C 18:0)

0

ED

Fatty acid content (g 100 g )

(C 18:1 n7)

2

0 -1

Oleic (C 18:1 n9)

1

M

Diet

AN

Table 4 Fatty acid content and fatty acid profile in excreta of rats fed control diets and diets supplemented with amidated alginate

a

6.95

b

5

10

10

20

b

SEM

40 c

6.00c

1.24a

1.04b

5.71

1.17a 42.71

4

41.97

b

39.71

0.23 0.04 c

0.91

1.36a

1.34a

2.06b

1.40a

0.09

36.32a

23.74b

25.36bc

26.20c

0.93

1.27

a

1.37

ac

10.15

a

16.09

12.83

a

9.98

b

0.84

ac

a

b

1.19

a

1.68

14.76

b

10.27

b

0.06

15.30

b

0.45

10.98

b

0.26

1.02

SFA

44.62a

61.25b

59.32bc

57.38c

1.20

a

b

bc

29.80

c

0.93

15.78 11.94 11.72 12.83 in the same row with different superscripts differ significantly at P < 0.05

b

0.34

PUFA abc Values

39.61

a

26.81

b

14

28.97

0.98

a

Linolenic (C 18:3)

MUFA

0.64

c

c

b

0.04

Figure 1