1. Plant stanol and sterol esters - mechanism and safety issues -
Jogchum Plat
Department of Human Biology
Nutrition and Toxicology Research Institute Maastricht
Maastricht University
The Netherlands

2. Topics
Mechanism (effects on cholesterol metabolism) 2. Safety aspects * Atherogenicity of plant sterols (and stanols?) * Red blood cell fragility

3. Topics
Mechanism (effects on cholesterol metabolism) 2. Safety aspects * Atherogenicity of plant sterols (and stanols?) * Red blood cell fragility

4. Plant sterols and stanols lower absorption from the intestinal lumen
Cholesterol
50%
Cholesterol plus
sterols / stanols
80%
20%

5. Plant sterols lower micellar cholesterol in the intestinal lumen of rats
Rats were fed diets for one week enriched with cholesterol (C) or
cholesterol + sitosterol (C+S). Analysis 2 hours after last meal
Aqueous (micel) phase
cholesterol
sitosterol
C C+S
C C+S
Ikeda et al. Biochim Biophys Act, 1983

6. Frequency study: Effect of intake moments
of plant stanol esters over the day
Control
Breakfast Lunch Diner
no stanols
2.5 g stanols
no stanols
Once
per day
0.4 g stanols
0.8 g stanols
1.3 g stanols
Three
times
per day

7. Consumption frequency does not change the effects of plant stanol esters
mmol/L
% change * * * *
LDL cholesterol
LDL cholesterol
Control
Once a day
2.5 g stanols
Three times a day
2.5 g stanols
* P<0.001 vs control group
Plat J et al. Eur J Clin Nutr, 2000

8. Plant stanol esters are already effective when consumed all at
Conclusion
Plant stanol esters are already effective when consumed all at
once (2.5 g/day) at lunch
This is the basis behind
the currently available
one-shot yogurt drinks

9. Is the effect on micelles in the intestinal lumen really underlying the cholesterol-lowering effect?
bile salt
Lipid core
Hypothesis
Plant sterols and stanols not only lower micellar cholesterol
concentrations, but also affect cholesterol metabolism
inside the enterocytes

10. Two plant sterol / stanol families
Cholesterol HO
4-desmethylsterols HO
Sitosterol HO
Campesterol
4,4-dimethylsterols HO
alpha-Amyrin
saturation
saturation HO
Sitostanol HO
Campestanol HO
Lupeol

11. Different types of plant sterols, different effects on LDL cholesterol concentrations
mmol/L
LDL cholesterol
% change * *
Zouden het “classes” of “types” zij of is dat allemaal hetzelfde?? * *
Control
Plant stanols
Plant sterols
Sheanut oil
(4,4-dimethylsterols)
(4-desmethylsterols)
* P<0.05 vs control group
Westrate et al. Eur J Clin Nutr, 1998

12. Both classes of plant sterol lower micellar cholesterol incorporation
4-desmethyl sterols
4,4-dimethyl sterols
% cholesterol incorpotared
Into mixed micelles
Cholesterol
Sitosterol
Sitostanol
a-amyrin
Lupeol
Plat et al. J Lpid Res 2005

13. Cholesterol homeostasis in enterocytes
chylomicrons
B48 CE
mesentheric lymph /
blood compartment
nucleus
enterocytes
cholesterol
Plant stanols
microvilli
brush border
membrane
ABC transporter
NPC1L1
NPC1L1
intestinal lumen
cholesterol
mixed micelles
mixed micelles

14. % Change ABCtransporter
Plant stanols / sterols increase ABC transporter expression in enterocytes
4-desmethyl
plant sterols
% Change ABCtransporter
expression
4,4-dimethyl
plant sterols
Legenda aangepast zodat de x-as wordt onderbroken en niet de legenda. Zie maar wat je mooier vindt
Titel aangepast
Sitosterol
Lupeol
Sitostanol
Cholesterol
Alpha-amyryn
Plat et al. J Lipid Res 2005

15. 4-desmethyl plant sterols / stanols activate LXR
LXRa
LXR
signal
signal
(nM)
(nM)
Cholesterol
Sitosterol
Campesterol
GW683965A
DMSO
LXRa
EC50 max signal
LXRb
EC50 max signal
Bij NWO kreeg Mark Boekschoten veel vragen over de karaketeristieken van de promotor (humaan of muis, lengte etc). Ben jij daarvan op de hoogte?
Cholesterol
Sitosterol
Campesterol
Sitostanol
Campestanol
GW683965A 72 42 43
136
122
197 75 76 86 52 54
104 63 26 28
110
124 41 71 80 77 38 45
Plat et al. J Lipid Res 2005

16. 4,4-dimethyl plant sterols do not activate LXR
LXRa
LXR
signal
signal
(nM)
(nM)
Lupeol
Alpha-amyrin
GW683965A
DMSO
LXRa
EC50 max signal
LXRb
EC50 max signal ND 77
Alpha-amyrin
lupeol
GW683965A ND
197 ND
104 ND 41
Plat et al. J Lipid Res 2005

17. Conclusion
Plant sterols and stanols lower intestinal cholesterol absorption, a process in which local activation of LXR target genes (cholesterol transporters) in the intestine plays a prominent role
Tekst iets aangepast. Zeg je hier niet hetzelfde als in de vorige slide??

18. Effects of plant stanols on whole body cholesterol metabolism
LIVER
VLDL
cholesterol
synthesis +
Remnant
receptor
IDL
LDL
receptor
Less cholesterol
enters the liver
by chylomicrons
LDL ?

19. Experimental design control margarine (n=17) low erucic acid
rapeseed (LEAR)
margarine
and shortening
plant stanol ester margarine (n=34)
- 4
(weeks) 8
isolation of PBMCs
Change

20. LDL receptor mRNA concentrations are significantly increased by dietary plant stanol esters
 LDLr mRNA
copies / mg total RNA *
* P<0.001 vs control group
Control group 
Plant stanol ester group
Plat and Mensink, FASEB J 2001

21. Are LDL receptor protein levels also increased ?
mRNA
LDL receptor
protein
- Wordt de LDL receptor posttranscriptional gemodificeerd?
- Kun je niet beter zeggen dat er geen 1:1 relatie is tussen mRNA en LDL-protein

22. LDL cholesterol reduction is correlated
with LDL receptor protein expression on monocytes
r=-0.492
P=0.0004; N=51
200
 LDLreceptor expression
100
-100
-1.2
-0.8
-0.4
0.4
0.8
 LDL cholesterol change (mmol/L)
Plat and Mensink, FASEB J 2001

23. Summary: effects on cholesterol metabolism
LIVER
cholesterol
synthesis +
VLDL
Remnant
receptor
IDL
chylomicrons
B48 CE
LDL
receptor
LDL +
cholesterol
ABC
transporter
chol
INTESTINE

24. Topics
Mechanism (effects on cholesterol metabolism) 2. Safety aspects * Atherogenicity of plant sterols (and stanols?) * Red blood cell fragility

25. Topics
Mechanism (effects on cholesterol metabolism) 2. Safety aspects * Atherogenicity of plant sterols (and stanols?) * Red blood cell fragility

26. High sitosterol is associated with a 2-fold
increased risk beyond high LDL cholesterol in men
Hazard ratio for coronary events
sitosterol (mmol/L)
LDL cholesterol (mmol/L)
Data from PROCAM
Assmann et al, NMCD 2006

27. Particularly statin treatment elevates
serum plant sterol concentrations
4S-study:
Subjects with high intestinal
cholesterol absorption and low
synthesis at baseline (Q4) did
not show a decrease in mortality
despite a similar cholesterol reduction. RR
Q1 0.62
Q2 0.66
Q3 0.75
Q4 1.16
Low abs / high synth
high abs / low synth
Is the increased plant sterol concentration
during statin treatment the reason why
mortality does not decrease in Q4 ?
Cholesterol mmol/L
weeks
Q1 placebo
Q1 statin
Q4 statin
Q4 placebo
Q1 placebo
Q1 statin
Q4 statin
Q4 placebo
weeks
Change campesterol (chol stand)
Miettinen et al. ATVB 2002, BMJ 1998

28. Intervention study in LDLr (+/-) mice
Run-in period
Experimental period
Western type diet (N=12)
Western type diet + 1% plant stanols (N=12)
Western type diet + 1% plant sterols (N=12)
Western type diet + atorvastatin (0.0025%)
+ atorvastatin (0.0025%)
Western type diet
(N=72)
0 Weeks
Bloodsampling x x x x x
Leasion typing x

29. Adding plant sterols or stanols to atorvastatin
treatment further lowers serum total cholesterol
Total cholesterol
(mmol/L)
Weeks
Plat et al. J Lipid Res 2006, in press

30. Effects on serum plant sterol concentrations
Control
Stanol
Sterol
Statin +
statin *
Sitosterol
(mmol/mmol cholesterol)
11-fold 
vs stanol
+ statin
Control
Stanol
Sterol
Statin +
statin *
Campesterol
(mmol/mmol cholesterol)
4-fold 
vs stanol
+ statin

31. Plant stanols and sterols (alone or combined with statins) lower lesion size in female heterozygous LDL-rec (+/-) deficient mice
Lesion area
(mm2) * * * *# *#
* vs control
# vs statin alone
Control
Stanol
Sterol
Statin
Stanol +
statin
Sterol +
statin

32. Conclusion
In heterozygous LDLreceptor deficient mice fed a western type diet:
- atorvastatin treatment alone as well as in combination with a plant sterol
enriched diet elevates serum plant sterol concentrations up to
11-fold (sitosterol)
atherosclerotic lesions size plus severity are improved, despite increased
serum plant sterol concentrations
This does not confirm the suggested atherogenicity
of plant sterols (or stanols)

33. Topics
Mechanism (effects on cholesterol metabolism) 2. Safety aspects * Atherogenicity of plant sterols (and stanols?) * Red blood cell fragility

34. Plant sterols / stanols and red blood cells
Red blood cell abnormalities in sitosterolemic patients
Stroke-prone spontaneously hypertensive rats have a shortened life span after plant sterol or plant stanol consumption (Ratnayake, 2000)
Question
Are elevated plant sterol or stanol concentrations in red blood cells harmfull?

35. Study design 45 statin users 4 weeks control ‘light’ margarine
16 weeks experimental ‘light’ margarine (3 groups of n=15)
30 gram of a 8 % plant sterol/stanol ‘light’ margarine (2.5 g plant sterol/stanol)
Osmotic fragility was measured in week 3 (baseline) and week 19
De opzet van deze studie ziet er als volgt uit. 45 Statine gebruikers hebben de studie inmiddels afgerond en naar type 2 diabeten zijn we nog hard op zoek, inmiddels zijn er 9 diabeten in de studie. Gedurende de eerste 4 weken wordt een controle halvarine gegeven waaraan dus geen plant sterolen of stanolen zijn toegevoegd. Na deze 4 weken zijn de proefpersonen verdeeld over 3 groepen. 1 Groep blijft de controle halvarine gebruiken, 1 groep krijgt een halvarine met plant sterolen en de ander met plant stanolen. Deze experimentele halvarine werd gebruikt gedurende 16 weken. De proefpersonen gebruikten per dag 30 gram halvarine, wat gelijk staat aan 2.5 g plant sterolen of stanolen. Dit betekent dat ongeveer 6 boterhammen/crackers/beschuiten met halvarine per dag moesten worden genuttigd. Gedurende het onderzoek werd aan het eind van de run in periode, 2 weken na het begin van de interventie periode en aan het eind van de interventieperiode bloed afgenomen.
De Jong, Plat and Mensink, Eur J Clin Nutr 2006

36. Hemolysis
Eerst zal ik uitleggen hoe we de kwetsbaarheid van rode bloedcellen hebben gemeten en vervolgens zal ik onze resultaten laten zien. In deze figuur is te zien hoe een rode bloedcel zich gedraagt in verschillende vloeistiffen. Bij een isotone vloeistof gebeurt er niets met de bloedecel, waneer de vloeistof “zout” is trekt al het vocht uit de bloedcel en klapt hij in. Wanneer de vloeistof juist “zoutloos” is trekt vocht de bloedcel in en zwelt deze op tot een bepaald volume en knapt dan uit elkaar. Dit wordt hemolyse genoemd. De hoeveelheid hemolyse neemt dus toe naarmate de zoutconcentratie van de omgeving afneemt. De mate van hemolyse (roodheid door het hemoglobine wat vrij komt) kunnen we kwntificeren.

37. Methods of the osmotic fragility measurement
RBC isolation
5 different saline concentrations (0.1, 0.35, 0.4, 0.45, 0.5%)
Spectrophotometer (540 nm)
We isoleren dus de rode bloedcellen uit the bloed van een proefpersoon, brengen deze in 5 oplossingen met verschilende zoutconcentraties en meten de hoeveelheid hemolyse.

38. No change of osmotic fragility in the control group
De volgende drie dia’s geven de uitkomsten van de kwetsbaarheid oftewel fragiliteit van rode bloedcellen voor de drie verschillende groepen. De rode lijn geeft de baseline meting, dus aanhet eind van de run in periode. De gele lijn geeft de meting aan het eind van de interventieperiode weer. In de controle groep is te zien dat er niets veranderd voor en na interventie.
De Jong, Plat and Mensink, Eur J Clin Nutr 2006

39. No change of osmotic fragility in the plant sterol and stanol groups
Ditzelfde geldt voor de plant sterol groep.
De Jong, Plat and Mensink, Eur J Clin Nutr 2006

40. Conclusion
Plant sterols and stanol esters, at a daily intake of 2.5 g do not change osmotic fragility of the red blood cell

41. Topics
Mechanism (effects on cholesterol metabolism) 2. Safety aspects * Atherogenicity of plant sterols (and stanols?) * Red blood cell fragility