Metabolic Effects of a Hydrophobic Alginate Derivative and Tetrahydrolipstatin in Rats Fed a Diet Supplemented with Palm Fat and Cholesterol.

The effects of octadecylamide of alginic acid (amidated alginate) and tetrahydrolipstatin on serum and hepatic cholesterol, and the faecal output of fat and sterols, were investigated in rats. Amidated alginate is a sorbent of lipids, tetrahydrolipstatin is an inhibitor of pancreatic lipase. Rats were fed diets containing cholesterol and palm fat at 10 and 70 g/kg, respectively. Palm fat was provided by coconut meal. Amidated alginate at 40 g/kg diet significantly decreased serum total cholesterol, low-density lipoprotein and hepatic cholesterol, and hepatic lipids and increased the faecal output of fat and coprostanol. Tetrahydrolipstatin at 300 mg/kg diet significantly decreased low-density lipoprotein cholesterol and hepatic lipids and increased the faecal output of fat. The intake of feed was not significantly influenced; however, the weight gains in rats fed amidated alginate were lower than in rats of the control group. Both amidated alginate and tetrahydrolipstatin modified the fatty acid profile in excreta lipids. Concentrations of saturated fatty acids were decreased and those of unsaturated fatty acids increased. Despite different modes of action, amidated alginate and tetrahydrolipstatin were equally efficient in removing the dietary fat from the body.


Introduction
Overweight is the risk factor for covid-19 and several chronic diseases (coronary heart disease, cancer, hypertension, diabetes type 2). Classical treatments for overweight such as dieting, behavioural modification and exercise, often fail; thus, there is the need for drugs and agents efficient in the treatment of overweight. New drugs are inhibitors of food intake which reduce hunger perception, inhibitors of nutrient absorption, and drugs, which increase energy expenditure 1. Tetrahydrolipstatin In rats fed high fat diet containing corn oil THL at 200 mg/kg significantly decreased serum concentration of triglycerides, total and LDL cholesterol, but increased concentration of HDL cholesterol 7. The authors, however, did not measured faecal concentration of fat, cholesterol and daily faecal 3 output of fat and sterols. The information about the effect of diets on the fatty acid profile of faecal lipids is absent in the available literature. Thus, the aim of the present study was to compare the effect of THL and amidated alginate on the faecal output of fat, sterols, and fatty acid profile of faecal lipids in rats fed a diet supplemented with cholesterol and palm fat. We tested a hypothesis that the effects of amidated alginate and THL may be similar despite different mode of action. The alginate obtained from the supplier and amidated alginates were characterized in order to obtain information which may be important in the present experiment.

Animals and diets
Twenty-one 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 daily photoperiod cycle at a temperature of 22 ± 1 °C. All diets were supplemented with cholesterol at 10 g/kg, and with coconut meal at 124 g/kg. The coconut meal containing 56.5% fat was purchased in a local shop with healthy food. Diets contained 10.9% of fat, including cholesterol. After 4 weeks of adaptation of rats to new environment, the rats were randomly divided into three groups of 7 rats each.  The experiment duration was 3 weeks, and then, the rats were sacrificed by decapitation after anaesthesia by the inhalation of isoflurane (Nicholas Piramal India Ltd., London, U.K.). The rats received 4 g of feed 4 h before they were sacrificed 14.

Materials and reagents
The sodium salt of alginic acid, a low-viscosity product A1112, from brown algae, was obtained in a dry powder form from Sigma-Aldrich (Prague, Czech Republic). THL (Orlistat), a certified reference material (PHR 1445), microcrystalline cellulose and n-octadecylamine were purchased from Sigma-Aldrich.

Preparation of amidated alginate
6 Alginic acid was prepared by washing the sodium alginate with acidified ethanol, pure ethanol and acetone, followed by air drying. Alginic acid was esterified with methanol in the presence of sulfuric acid.
The solid reaction product was filtered, washed with ethanol and acetone, and then dried in air. The methyl ester was amino-dealkoxylated with n-octadecylamine 15.

Molecular weight assay
The reaction with organic dinitroderivatives is based on the ability to reduce dinitrosalicylic acid by reducing polysaccharide groups 17. The colour intensity was measured using a spectrophotometer at a wavelength of 545 nm. Galacturonic acid was used for the calibration.
The degree of polymerization and molecular weight were calculated from the absorbance of the polysaccharide sample.

Analysis of mannuronic to galacturonic acid ratio in alginate
Alginate was hydrolysed using trifluoracetic acid as described by Wang

Other analyses
The fatty acid (FA) profile of coconut meal, fat content in coconut meal, and analyses of ST-1 diet

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 standard deviation.
Significant differences (p < 0.05) were identified using Tukey´s test. The Pearson correlation coefficient was used as a measure of the dependence between pairs of observations.

Results
Average initial and final body weights of rats were 238.7 g and 272.3 g, respectively. Body weights of rats and the feed intake did not differ among treatment groups. Weight gain of rats fed amidated alginate was significantly lower than that of control rats (Table 2).

Characterization of palm fat and coconut meal
In coconut meal the main fatty acid was myristic acid followed by palmitic acid and capric acid. Oleic was the main unsaturated FA present in coconut meal (Table 4). Coconut meal contained 56.5% fat determined by extraction with petroleum ether. The HPLC analysis showed that coconut meal contained triacylglycerols expressed as tripalmitin at 623 mg/g. In total 35 fatty acids were assayed.  acids was decreased and that of unsaturated fatty acids was increased (Table 6). Additives Nutrient" concluded that there is no safety concern for the use of pectin and amidated pectin for the general population and that there is no need for "Acceptable daily intake" 23.

Effects of amidated alginate and tetrahydrolipstatin on serum and hepatic parameters and faecal output of fat and sterols in rats
Alginate is a natural polysaccharide, the composition of which depends on the algae species. The Bile acid synthesis in rat hepatocytes may be regulated by the availability of cholesterol for cholesterol 7α-hydroxylase 28.
Amidated alginate and THL increased the faecal loss of fat, the mode of action of both agents, however was different. Amidated alginate is a non-specific sorbent of lipids, THL inhibits triacylglycerol hydrolysis which is the prerequisite of the lipid absorption. Both amidated alginate and THL decreased concentration of saturated fatty acids in excreta of rats and increased concentration of monounsaturated and polyunsaturated fatty acids. This may be related to the fact that unsaturated lipids are more easily incorporated into mixed micelles in the intestine. It is possible, however, that in amidated alginate-fed rats also lipophilic vitamins may be partially lost in faeces.
It follows from data in Table 5