Deux approches ont été mises au point et utilisées afin de mieux comprendre les facteurs nutritionnels, physiopathologiques et génétiques intervenant dans la grande variabilité d'absorption du cholestérol chez l'homme (30-80%) :- le dosage de marqueurs plasmatiques d'absorption et de synthèse du cholestérol (METHODE INDIRECTE), qui s'applique aux grandes cohortes.- le dosage simultané de deux isotopes stables du cholestérol utilisés comme traceurs (METHODE DIRECTE), qui est la méthode de référence pour déterminer le taux d'absorption du cholestérol alimentaire.Nous avons déterminé, par la méthode indirecte, des interactions gène-régime-sexe, dans une population mixte présentant un risque cardiovasculaire modéré.On a montré qu'en modifiant les habitudes alimentaires, le statut d'absorption du cholestérol d'un sujet n'était pas modifié après 3 mois de régime méditerranéen, quel que soit le sexe. De plus la diminution du cholestérol circulant (LDL-C) induite par le régime de type méditerranéen était plus marquées chez les faibles absorbeurs de cholestérol. Le statut d'absorbeur est donc stable dans le temps et sous différents régimes, mais il module la réponse au régime, illustrant l'importance de ce statut dans la prise en charge nutritionnelle personnalisée des sujets. Nous avons aussi établi une interaction gène-régime sur le polymorphisme génétique du gène de la microsomal triglyceride transfer protein (-493G/T). Ce polymorphisme module le niveau d'absorption du cholestérol chez les femmes sous un régime occidental et cet effet est aboli sous un régime méditerranéen.La méthode indirecte présente cependant des limites. Aussi avons-nous mis au point une méthode de dosage directe originale, basée sur l'abondance relative des isotopomères du cholestérol, le but étant de mesurer simultanément les marqueurs directs et indirects et de comparer les deux méthodes en vue de l'application systématique de la méthode indirecte dans différentes études chez l'homme. / Two approaches were used to better understand nutritional, physio-pathological and genetic factors intervening in the great inter-individual variability of cholesterol absorption in humans (30-80%) : - INDIRECT METHOD consisting in the determination of both absorption and synthesis plasmatic markers of cholesterol, which is used in large scale studies; - a DIRECT METHOD by measuring simultaneaously two cholesterol stable isotopes, used as tracers. It is the "gold" method to determine the absorption percentage of dietary cholesterol. By using indirect method, we have shown gene-diet interactions according to sex in a population of men and women with moderate cardiovascular risks. Changing in dietary habits didn't alter cholesterol absorption statuts after 3 months of mediterranean type diet, whatever the sex. Moreover, the lowering of plasma LDL-cholesterol induced by mediterranean type diet was more marked in low-cholesterol absorbers. Thus, the cholesterol absorption status is stable over time and under differents regimes, but it modulates responsiveness to a dietary challenge. These findings illustrate the importance of cholesterol absorption status in personalized nutrition recommendations. Another finding is a gene-diet interaction in the -493G/T gene polymorphism of the microsomal triglyceride transfer protein. This polymorphism modulates the level of cholesterol absorption in women under a western diet, an effect abolished under a prudent mediterranean type diet. As indirect method shows limits, we established an original direct method, based on relative abundance of cholesterol isotopomers. Our goal is to measure simultaneously both direct and indirect plasmatic markers. Then, we could compare the two methods with the objective of applying systematically indirect method in different studies in humans.

The aim of this diploma thesis was to develop an electrochemical method for three phytosterols, namely stigmasterol β-sitosterol and ergosterol using voltammetry techniques on glassy carbon electrode and boron doped diamond electrode. The work builds on previous results, where it was shown that the electrochemical oxidation of phytosterols requires perchloric acid in acetonitrile. When this acid is used, a dehydration reaction occurs. Calibration dependences were measured here for all phytosterols in an environment with a water content of 0.43%. The stability of the signal with increasing distilled water content was further investigated. Then the calibration dependences in the increased water content were measured again. If we want to use HPLC-type separation techniques, we must first find out how the phytosterols will behave in this environment, and therefore the signal stability on the buffer content was investigated. The buffers used were phosphate and acetate buffer. Subsequently, the concentration dependence was measured in an environment with a higher content of phosphate buffer. Furthermore, the possibility of detecting two phytosterols at once was investigated. Key words: dehydration, glassy carbon electrode, boron doped diamond electrode, oxidation, phytosterols, voltammetry

The supply of precursors from primary metabolism is often overlooked when engineering secondary metabolism for increased product yields. This is because precursor supply may be assumed to be non-limiting, and it is considered difficult to engineer primary metabolism, because control of carbon flow (flux) is generally distributed among most enzymes of the pathway. The aim of this thesis was to increase the production of sterols, part of the isoprenoid class of secondary metabolites, in tobacco (Nicotiana tabacum) Bright Yellow 2 (BY-2) cell cultures. This was achieved by genetically engineering increased activity of mitochondrial citrate synthase, an enzyme of the tricarboxylic acid (TCA) cycle that is involved in the provision of cytosolic acetyl coenzyme A, the primary metabolite precursor to sterols. Metabolic flux analysis revealed that citrate synthase exerts significant control over cyclic TCA cycle flux in BY-2 cells and suggested that increasing the activity of downstream enzymes within secondary metabolism could lead to a further redirection of TCA-cycle-derived precursors into sterol biosynthesis. Attempts were made to achieve this by genetically engineering increased activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), a key enzyme of secondary metabolism involved in sterol biosynthesis. Consistent with previous research, transgenic lines had increased sterol levels. However, the high sterol phenotype was unstable, and attempts to co-express HMGR and citrate synthase genes were unsuccessful. The thesis demonstrates that increasing the provision of precursors to secondary metabolites can result in increased yields of those secondary metabolites but suggests that in most cases the activity of enzymes within secondary metabolism has a greater effect on those yields. It also reveals that single enzymes can exert significant control of flux within primary metabolism, although the control exerted by specific enzymes probably changes with the demands placed on metabolism.

Phytosterols have been shown to be effective in reducing serum cholesterol levels in numerous human clinical studies and regular consumption is recommended as part of therapeutic lifestyle changes aimed at reducing low density lipoprotein (LDL-C) in the treatment of hyperlipidaemia, a risk factor for cardiovascular disease. Fat based spreads have been shown to be a very successful vehicle for delivery of plant sterols, readily accepted by consumers and efficacious in reducing cholesterol levels. Alfa One™ Rice Bran Oil (RBO) spread is a new product entering into the market place. It is derived from rice bran oil and contains high levels of unsaponifiable material rich in phytosterols, triterpene alcohols, ferulic acid esters ([gamma]-oryzanol) and vitamin E isomers. As such it may have the potential to lower serum cholesterol levels when consumed on a daily basis. In order to establish the effectiveness of Alfa One™ Rice Bran Oil (RBO) spread compared with Flora pro-activ® margarine, a well established brand of plant sterol margarine already proven to lower cholesterol, a randomised double blind cross-over human clinical trial over 12 weeks was conducted. The study was divided into two treatment arms. The first arm of the study was to determine whether Alfa One™ RBO spread (containing 1.5% plant sterols) could lower total and LDL cholesterol levels to a greater extent than standard Flora margarine (containing no plant sterols) or Flora Pro-activ® margarine (containing 8% plant sterols). The second study arm tested the proposition that daily consumption of Alfa One™ Rice Bran Oil (RBO) spread in conjunction with rice bran oil (containing 0.5% plant sterols) would lower total and LDL cholesterol to a greater extent than Alfa One™ RBO spread in isolation and more than Flora margarine in conjunction with sunflower oil. Eighty mildly hypercholesterolaemic individuals (total cholesterol [greater than or equal to] 5 mmol/L and [less than or equal to] 7.5 mmol/L) were recruited and randomised into two groups of forty. Participants were asked to continue with their normal dietary pattern but to replace any margarine/butter/fat consumption with the trial products. One group of 40 were then assigned to the first treatment arm of the study (margarine-only group) and were randomised to consume 20 g (4 teaspoons) Alfa One™ RBO spread daily for 4 weeks, or 20 g Flora margarine daily for 4 weeks, or 20 Flora pro-activ® daily for 4 weeks. Phytosterol levels delivered in these amounts were: RBO margarine: 118mg phytosterol and 14 mg [gamma]-oryzanol; Flora proactiv® 1600 mg phytosterol; Flora margarine 0mg phytosterol. The second group of 40 were allocated to the second arm of the trial (margarine and oil group) and consumed 20 g Alfa One™ RBO spread and 30 ml rice bran oil (RBO) daily for 4 weeks, or 20 g Flora margarine and 30 ml sunflower oil daily for 4 weeks, or 20 g Alfa One™ RBO spread daily for 4 weeks, changing treatment at the end of each 4-week period. Phytosterol amounts delivered in these amounts were: RBO margarine: 118 mg phytosterol and 14 mg [gamma] oryzanol; RBO 222mg mg phytosterol, 150 mg [gamma] oryzanol. Each participant consumed all three treatments in a random order over a 12 week period. At baseline and following each 4 week intervention period, measurements were made of weight and blood pressure. Venous blood samples were collected for analysis of total cholesterol, low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), total cholesterol: HDL-C, triglycerides and plasma phytosterols. Three-day diet records from each individual were also collected for analysis of normal dietary intake. Results showed that compared to a standard Flora margarine, Alfa One™ RBO spread significantly reduced total cholesterol by 2.2% (P=0.045), total cholesterol:HDL by 4.1% (P=0.005) and LDL-C by 3.5% (P=0.016), but was not as effective overall as Flora Pro-activ® which reduced total cholesterol by 4.4% (P=0.001), total cholesterol:HDL by 3.4% (P=0.014) and LDL-C by 5.6% (P=0.001). Consumption of Flora margarine alone produced no significant decrease from baseline figures in any of the cholesterol parameters measured. Surprisingly, in group two, the addition of rice bran oil to the Alfa One™ RBO spread produced no differences in cholesterol levels. The reason for this unexpected result is being explored further. These results confirm that Alfa One™ RBO spread is effective in lowering serum cholesterol levels when consumed as part of a normal diet. Studies have shown that a 1% reduction in LDL-C can equate to a 2% decrease in coronary heart disease (CHD) risk thus suggesting that the 3.5% reduction demonstrated by Alfa One™ RBO spread in this study could be effective in reducing CHD risk as much as 6% in a mildly hypercholesterolaemic population.

Phytosterols have been shown to be effective in reducing serum cholesterol levels in numerous human clinical studies and regular consumption is recommended as part of therapeutic lifestyle changes aimed at reducing low density lipoprotein (LDL-C) in the treatment of hyperlipidaemia, a risk factor for cardiovascular disease. Fat based spreads have been shown to be a very successful vehicle for delivery of plant sterols, readily accepted by consumers and efficacious in reducing cholesterol levels. Alfa One™ Rice Bran Oil (RBO) spread is a new product entering into the market place. It is derived from rice bran oil and contains high levels of unsaponifiable material rich in phytosterols, triterpene alcohols, ferulic acid esters ([gamma]-oryzanol) and vitamin E isomers. As such it may have the potential to lower serum cholesterol levels when consumed on a daily basis. In order to establish the effectiveness of Alfa One™ Rice Bran Oil (RBO) spread compared with Flora pro-activ® margarine, a well established brand of plant sterol margarine already proven to lower cholesterol, a randomised double blind cross-over human clinical trial over 12 weeks was conducted. The study was divided into two treatment arms. The first arm of the study was to determine whether Alfa One™ RBO spread (containing 1.5% plant sterols) could lower total and LDL cholesterol levels to a greater extent than standard Flora margarine (containing no plant sterols) or Flora Pro-activ® margarine (containing 8% plant sterols). The second study arm tested the proposition that daily consumption of Alfa One™ Rice Bran Oil (RBO) spread in conjunction with rice bran oil (containing 0.5% plant sterols) would lower total and LDL cholesterol to a greater extent than Alfa One™ RBO spread in isolation and more than Flora margarine in conjunction with sunflower oil. Eighty mildly hypercholesterolaemic individuals (total cholesterol [greater than or equal to] 5 mmol/L and [less than or equal to] 7.5 mmol/L) were recruited and randomised into two groups of forty. Participants were asked to continue with their normal dietary pattern but to replace any margarine/butter/fat consumption with the trial products. One group of 40 were then assigned to the first treatment arm of the study (margarine-only group) and were randomised to consume 20 g (4 teaspoons) Alfa One™ RBO spread daily for 4 weeks, or 20 g Flora margarine daily for 4 weeks, or 20 Flora pro-activ® daily for 4 weeks. Phytosterol levels delivered in these amounts were: RBO margarine: 118mg phytosterol and 14 mg [gamma]-oryzanol; Flora proactiv® 1600 mg phytosterol; Flora margarine 0mg phytosterol. The second group of 40 were allocated to the second arm of the trial (margarine and oil group) and consumed 20 g Alfa One™ RBO spread and 30 ml rice bran oil (RBO) daily for 4 weeks, or 20 g Flora margarine and 30 ml sunflower oil daily for 4 weeks, or 20 g Alfa One™ RBO spread daily for 4 weeks, changing treatment at the end of each 4-week period. Phytosterol amounts delivered in these amounts were: RBO margarine: 118 mg phytosterol and 14 mg [gamma] oryzanol; RBO 222mg mg phytosterol, 150 mg [gamma] oryzanol. Each participant consumed all three treatments in a random order over a 12 week period. At baseline and following each 4 week intervention period, measurements were made of weight and blood pressure. Venous blood samples were collected for analysis of total cholesterol, low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), total cholesterol: HDL-C, triglycerides and plasma phytosterols. Three-day diet records from each individual were also collected for analysis of normal dietary intake. Results showed that compared to a standard Flora margarine, Alfa One™ RBO spread significantly reduced total cholesterol by 2.2% (P=0.045), total cholesterol:HDL by 4.1% (P=0.005) and LDL-C by 3.5% (P=0.016), but was not as effective overall as Flora Pro-activ® which reduced total cholesterol by 4.4% (P=0.001), total cholesterol:HDL by 3.4% (P=0.014) and LDL-C by 5.6% (P=0.001). Consumption of Flora margarine alone produced no significant decrease from baseline figures in any of the cholesterol parameters measured. Surprisingly, in group two, the addition of rice bran oil to the Alfa One™ RBO spread produced no differences in cholesterol levels. The reason for this unexpected result is being explored further. These results confirm that Alfa One™ RBO spread is effective in lowering serum cholesterol levels when consumed as part of a normal diet. Studies have shown that a 1% reduction in LDL-C can equate to a 2% decrease in coronary heart disease (CHD) risk thus suggesting that the 3.5% reduction demonstrated by Alfa One™ RBO spread in this study could be effective in reducing CHD risk as much as 6% in a mildly hypercholesterolaemic population.

Phytosterols have been shown to be effective in reducing serum cholesterol levels in numerous human clinical studies and regular consumption is recommended as part of therapeutic lifestyle changes aimed at reducing low density lipoprotein (LDL-C) in the treatment of hyperlipidaemia, a risk factor for cardiovascular disease. Fat based spreads have been shown to be a very successful vehicle for delivery of plant sterols, readily accepted by consumers and efficacious in reducing cholesterol levels. Alfa One™ Rice Bran Oil (RBO) spread is a new product entering into the market place. It is derived from rice bran oil and contains high levels of unsaponifiable material rich in phytosterols, triterpene alcohols, ferulic acid esters ([gamma]-oryzanol) and vitamin E isomers. As such it may have the potential to lower serum cholesterol levels when consumed on a daily basis. In order to establish the effectiveness of Alfa One™ Rice Bran Oil (RBO) spread compared with Flora pro-activ® margarine, a well established brand of plant sterol margarine already proven to lower cholesterol, a randomised double blind cross-over human clinical trial over 12 weeks was conducted. The study was divided into two treatment arms. The first arm of the study was to determine whether Alfa One™ RBO spread (containing 1.5% plant sterols) could lower total and LDL cholesterol levels to a greater extent than standard Flora margarine (containing no plant sterols) or Flora Pro-activ® margarine (containing 8% plant sterols). The second study arm tested the proposition that daily consumption of Alfa One™ Rice Bran Oil (RBO) spread in conjunction with rice bran oil (containing 0.5% plant sterols) would lower total and LDL cholesterol to a greater extent than Alfa One™ RBO spread in isolation and more than Flora margarine in conjunction with sunflower oil. Eighty mildly hypercholesterolaemic individuals (total cholesterol [greater than or equal to] 5 mmol/L and [less than or equal to] 7.5 mmol/L) were recruited and randomised into two groups of forty. Participants were asked to continue with their normal dietary pattern but to replace any margarine/butter/fat consumption with the trial products. One group of 40 were then assigned to the first treatment arm of the study (margarine-only group) and were randomised to consume 20 g (4 teaspoons) Alfa One™ RBO spread daily for 4 weeks, or 20 g Flora margarine daily for 4 weeks, or 20 Flora pro-activ® daily for 4 weeks. Phytosterol levels delivered in these amounts were: RBO margarine: 118mg phytosterol and 14 mg [gamma]-oryzanol; Flora proactiv® 1600 mg phytosterol; Flora margarine 0mg phytosterol. The second group of 40 were allocated to the second arm of the trial (margarine and oil group) and consumed 20 g Alfa One™ RBO spread and 30 ml rice bran oil (RBO) daily for 4 weeks, or 20 g Flora margarine and 30 ml sunflower oil daily for 4 weeks, or 20 g Alfa One™ RBO spread daily for 4 weeks, changing treatment at the end of each 4-week period. Phytosterol amounts delivered in these amounts were: RBO margarine: 118 mg phytosterol and 14 mg [gamma] oryzanol; RBO 222mg mg phytosterol, 150 mg [gamma] oryzanol. Each participant consumed all three treatments in a random order over a 12 week period. At baseline and following each 4 week intervention period, measurements were made of weight and blood pressure. Venous blood samples were collected for analysis of total cholesterol, low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), total cholesterol: HDL-C, triglycerides and plasma phytosterols. Three-day diet records from each individual were also collected for analysis of normal dietary intake. Results showed that compared to a standard Flora margarine, Alfa One™ RBO spread significantly reduced total cholesterol by 2.2% (P=0.045), total cholesterol:HDL by 4.1% (P=0.005) and LDL-C by 3.5% (P=0.016), but was not as effective overall as Flora Pro-activ® which reduced total cholesterol by 4.4% (P=0.001), total cholesterol:HDL by 3.4% (P=0.014) and LDL-C by 5.6% (P=0.001). Consumption of Flora margarine alone produced no significant decrease from baseline figures in any of the cholesterol parameters measured. Surprisingly, in group two, the addition of rice bran oil to the Alfa One™ RBO spread produced no differences in cholesterol levels. The reason for this unexpected result is being explored further. These results confirm that Alfa One™ RBO spread is effective in lowering serum cholesterol levels when consumed as part of a normal diet. Studies have shown that a 1% reduction in LDL-C can equate to a 2% decrease in coronary heart disease (CHD) risk thus suggesting that the 3.5% reduction demonstrated by Alfa One™ RBO spread in this study could be effective in reducing CHD risk as much as 6% in a mildly hypercholesterolaemic population.

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Phytosterols have been shown to be effective in reducing serum cholesterol levels in numerous human clinical studies and regular consumption is recommended as part of therapeutic lifestyle changes aimed at reducing low density lipoprotein (LDL-C) in the treatment of hyperlipidaemia, a risk factor for cardiovascular disease. Fat based spreads have been shown to be a very successful vehicle for delivery of plant sterols, readily accepted by consumers and efficacious in reducing cholesterol levels. Alfa One™ Rice Bran Oil (RBO) spread is a new product entering into the market place. It is derived from rice bran oil and contains high levels of unsaponifiable material rich in phytosterols, triterpene alcohols, ferulic acid esters ([gamma]-oryzanol) and vitamin E isomers. As such it may have the potential to lower serum cholesterol levels when consumed on a daily basis. In order to establish the effectiveness of Alfa One™ Rice Bran Oil (RBO) spread compared with Flora pro-activ® margarine, a well established brand of plant sterol margarine already proven to lower cholesterol, a randomised double blind cross-over human clinical trial over 12 weeks was conducted. The study was divided into two treatment arms. The first arm of the study was to determine whether Alfa One™ RBO spread (containing 1.5% plant sterols) could lower total and LDL cholesterol levels to a greater extent than standard Flora margarine (containing no plant sterols) or Flora Pro-activ® margarine (containing 8% plant sterols). The second study arm tested the proposition that daily consumption of Alfa One™ Rice Bran Oil (RBO) spread in conjunction with rice bran oil (containing 0.5% plant sterols) would lower total and LDL cholesterol to a greater extent than Alfa One™ RBO spread in isolation and more than Flora margarine in conjunction with sunflower oil. Eighty mildly hypercholesterolaemic individuals (total cholesterol [greater than or equal to] 5 mmol/L and [less than or equal to] 7.5 mmol/L) were recruited and randomised into two groups of forty. Participants were asked to continue with their normal dietary pattern but to replace any margarine/butter/fat consumption with the trial products. One group of 40 were then assigned to the first treatment arm of the study (margarine-only group) and were randomised to consume 20 g (4 teaspoons) Alfa One™ RBO spread daily for 4 weeks, or 20 g Flora margarine daily for 4 weeks, or 20 Flora pro-activ® daily for 4 weeks. Phytosterol levels delivered in these amounts were: RBO margarine: 118mg phytosterol and 14 mg [gamma]-oryzanol; Flora proactiv® 1600 mg phytosterol; Flora margarine 0mg phytosterol. The second group of 40 were allocated to the second arm of the trial (margarine and oil group) and consumed 20 g Alfa One™ RBO spread and 30 ml rice bran oil (RBO) daily for 4 weeks, or 20 g Flora margarine and 30 ml sunflower oil daily for 4 weeks, or 20 g Alfa One™ RBO spread daily for 4 weeks, changing treatment at the end of each 4-week period. Phytosterol amounts delivered in these amounts were: RBO margarine: 118 mg phytosterol and 14 mg [gamma] oryzanol; RBO 222mg mg phytosterol, 150 mg [gamma] oryzanol. Each participant consumed all three treatments in a random order over a 12 week period. At baseline and following each 4 week intervention period, measurements were made of weight and blood pressure. Venous blood samples were collected for analysis of total cholesterol, low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), total cholesterol: HDL-C, triglycerides and plasma phytosterols. Three-day diet records from each individual were also collected for analysis of normal dietary intake. Results showed that compared to a standard Flora margarine, Alfa One™ RBO spread significantly reduced total cholesterol by 2.2% (P=0.045), total cholesterol:HDL by 4.1% (P=0.005) and LDL-C by 3.5% (P=0.016), but was not as effective overall as Flora Pro-activ® which reduced total cholesterol by 4.4% (P=0.001), total cholesterol:HDL by 3.4% (P=0.014) and LDL-C by 5.6% (P=0.001). Consumption of Flora margarine alone produced no significant decrease from baseline figures in any of the cholesterol parameters measured. Surprisingly, in group two, the addition of rice bran oil to the Alfa One™ RBO spread produced no differences in cholesterol levels. The reason for this unexpected result is being explored further. These results confirm that Alfa One™ RBO spread is effective in lowering serum cholesterol levels when consumed as part of a normal diet. Studies have shown that a 1% reduction in LDL-C can equate to a 2% decrease in coronary heart disease (CHD) risk thus suggesting that the 3.5% reduction demonstrated by Alfa One™ RBO spread in this study could be effective in reducing CHD risk as much as 6% in a mildly hypercholesterolaemic population.