Efficiency of intestinal cholesterol absorption in humans is not related to apoE phenotype
Human apolipoprotein E has three major isoforms (ApoE2, ApoE3 and ApoE4), it is a polymorphic and translates into three common alleles (ε2, ε3 and ε4), all together it forms six phenotypes. These alleles show its activity against total cholesterol, LDL-cholesterol, apoB, and apoE concentrations. There has been several mechanism for different isoforms of apoE for its activity on plasma concentration and some of them are:- 1)Receptor-binding affinity of the different apoE containing lipoproteins, 2)Dietary fat clearance, 3)Difference in the clearance of LDL apoB, 4)Efficiency of intestinal cholesterol absorption. One ε4 allele in subject compared with ε3 allele in other subject homozygous, showed higher intestinal cholesterol absorption efficiency in ε4 allele. The apoE3 in three subjects compared with E2 allele homo- or heterozygous, showed higher cholesterol absorption in apoE3. This study was carried out in one subject homozygous for ε4 allele and four patients for apoE2 isoform, which include one homozygous for ε2 allele. Miettinen et al. performed other study in subject with apoε2, apoε3 and apoε4 alleles, it result showed that apoε3 and apoε4 had a significant higher absorption than that of apoε2 alleles. At low cholesterol diet, the difference in the above was not seen. The cholesterol absorption efficiency was not studied symmetrically with that of apoE phenotype. Therefore they decided to measure the cholesterol absorption efficiency in the subject homozygous with cholesterol and bile acid synthesis for the ε4 and ε2 alleles. In their further study they divided there subject into normal plasma cholesterol and triglycerides and subjects with familial III hyperlipoproteinemia. They also measured plant sterol campesterol in plasma for all subjects side by side. They came to know that there is direct relationship between dietary campesterol and cholesterol absorption and indirect relationship between the ratio of plasma campesterol to cholesterol to that of the rate of cholesterol absorption.
MATERIALS AND METHODS
Experimental design and subjects:
They have performed this study to compare the cholesterol absorption efficiency with apoE2/2 in subjects and with that of apoE4/4 in other subjects. The subjects participated in study were 6 normolipemic volunteers for E2/2, 6 patients for E2/2 with type III hyperlipoproteinemia and 8 subjects for E4/4 including 6 hypercholesterolemia patients. All the subjects which were selected did not had a history of excessive alcohol intake, diabetes, or other endocrine disorders, renal disease, or diseases of the liver or gastrointestinal tract. These subjects were not given lipid-lowering drugs prior to the 6 weeks of the study. They have measured the cholesterol absorption and fecal excretion of neutral and acidic sterols during the first week of the study. They collected blood samples and analysed the plasma lipids after the overnight fast of the subjects. They have also analysed the noncholesterol at the start and end of this week.
They have performed this experiment but the week point of this experimental design was that they have take only 20 subjects in the study, which are very less to get the accurate result. If they would have taken more number of subjects for the study than they would got more efficient and accurate result. The strong point of this experiment was that the subjects were not give the lipid-lowering drug to calculate the proper absorption of cholesterol from the intestine, if they have given this drug than the absorption of the cholesterol would have decreased and could not find the exact cholesterol absorption. The other strong point of the experimental design was that they have selected the subjects which were selected were free from diseases and alcohol intake.
They have determine apoE phenotypes by using isoelectric focusing technique for apolipoproteins with immunoblooting and the strong point of this technique is that it is more advantages than any other technique. This method gives a precast immolized pH gradient and it does not consume time in separation of lipoproteins prom plasma, if we r using this technique than there is no need for pertreatment with neuraminidase and it shows good result at it is highly sensitive for visualization.( Golaz O et al. 1995)
They have measured the total plasma cholesterol and triglycerides from the bllod samples which were collected from the subject after they have been in fast for overnight of the study week at the beginning and the end by using commercial kit. The strong point of the method was that it measured accurately and more efficient than other methods.( RussellWarnick et al. 2001)
They have used a modified gas liquid chromatography method for the measurement of concentration of cholesterol and noncholesterol sterols in plasma. The strong point of this method is that it seperate the component of the plasma and measure the concentration of cholesterol and non cholesterol sterols and it give good accuracy than any other method.( Pavia et al. 2006).
Isotope feeding method was used for the measurement of absorption of cholesterol from the stomach and the fecal ratio of deuterium-labeled cholesterol and its bacterial degradation products coprostanol, coprostanone and deuterium-labeled sitostanol were calculated by fractional cholesterol absorption. Gas liquid chromatography-mass spectrometry was used to calculate the ratio of deuterium-labelled cholesterol and sitostanol in the capsules. At last the cholesterol absorption percentage was determined from the complete loss of cholesterol in the small intestine during the passage compared with sitostanol. The strong point of this method is that it does not require intraduodenal intubation, and daily measurement but it requires only plasma and stool collection for analysis and the other is that the mass spectroscopy is used as a detector and give the accurate result.( Crouse et al. 1983).
They have collected the fecal samples from the cholesterol absorption for the measurement of the fecal excretion of neutral and acidic sterols, to measure this they have given the sitostanol to the subjects for the fecal recovery maker to that of stable isotope makers for 7 days. This was one of the strong point for the getting the fecal sample and then measuring the fecal excretion of neutral and acidic sterols with sitostanol was done by gas liquid chromatography. Secondly they have daily measured the facal excretion rates of neutral and acidic sterols with that of net cholesterol balance was calculated.
They have kept the record of dietary materials as dietary intake give to the subject for the calculation of the dietary cholesterol, protein, fat carbohydrate and fiber intake was calculated by computer program. The strong point of this was that they have kept record of all thing which were given to the subject but the weak point was that they have not mention the program name, which was used for the calculation.
In this study the result was given as mean ± SEM, which gives an average of the outcome. Student's t-test was used for calculating the difference in percentage of cholesterol absorption, diatery cholesterol intake, fecal excretion neutral and acidic sterols, cholesterol synthesis, plasma lipoproteins and plasma noncholesterol sterols. Linear regression and stepwise regression method was used for calculating the correlations of outcome. In this method, result showing the value of p<0.05 is said to be significant. All this statistical data were managed and analysed by using statistical software SPSS/windows. The strong point of this software is that it calculate the exact value from data by using different methods and here t-test was used, in this test p value less than 0.05 is said to be significant and greater than 0.05 is not considered significant.
The outcome of the studies in subjects with their clinical characteristics and plasma concentration of cholesterol and triglycerides showed that the body weight was greater in type III hyperlipoproteinemia with that of other two groups and that of body mass indexes were different in all groups. Plasma cholesterol and triglycerides was greater in subject with type III hyperlipoproteinemia than that of normolipemic subjects homozygous for ε2 alleles, for ε4 alleles total cholesterol and triglycerides was greater than homozygous. Normolipemic volunteers homozygous had lower plasma concentration than the latter subjects for apoE2. Result obtained by gas liquid chromatography and enzymatically showed the same total cholesterol level in the subjects.
The cholesterol absorption for three groups in subjects ranged from 27% to 53%. The cholesterol absorption was 38% as an averaged for all apoE2/2 with that of apoE4/4 was 41%, so there was not a much difference between the groups. There was also not much different between the cholesterol absorption in normolipemic apoE2/2volunteers and type III hyperlipoproteinemia patients. Energy intake was greater in normolipemic E2/2 subjects (2488±658kcal/day) with that of hyperlipedemic E2/2 (1968±662 kcal/day) and E4/4 (2006±440 kcal/day) carriers, protein intake and fiber (g/d) (ns) were lower in percentage of total calories (p < 0.05).cholesterol absorption was not dependent on dietary intake, body weight and age. Normolipemic E2/2 volunteers had greater dietary intake of cholesterol than that of other two groups. Synthesis of cholesterol and bile acid were same in all groups and positive relationship was seen between BMI and cholesterol synthesis (r = 0.626; p<0.005) and between body weight and cholesterol synthesis (r = o.574; p<0.01). from the given figure 1 below:
Type III hyperlipoproteinemia patients showed greater concentration of all noncholesterol sterols to that of normolipemic apoE2/2 carriers. Type III hyperlipoproteinemia also showed higher plasma concentration of lathosterol with that of subject of apoE4/4 alleles. All noncholesterol plasma sterols in homozygous subjects for ε4 was greater compared to normolipemic E2/2 volunteers but this result was not shown statistically significant. The three groups of subjects had no differences between the ratios of cholestanol, lathosterol and campesterol to that of cholesterol. In all subjects they found a significant correlation between the ratio of campesterol to cholesterol in plasma (r = 0.504; p<0.02) and the efficiency of cholesterol absorption from the above figure 1.
They have performed this study in human to know the role of the apoE on cholesterol absorption, so they have participated homozygous subject either for the ε2 or the ε4 allele. Further the apoE2 homozygous group selected were of two type 1) normocholesterolemic (six subjects) and 2) type III hyperlipoproteinemia (six subjects). They also compared noncholesterol sterols, bile acid and total synthesis in plasma. This was the first study ever performed in which cholesterol absorption was compared with homozygous subjects for the apoε2 or ε4 allele. This study showed good results than previous studies to find out the role of apoE on cholesterol absorption.
In this study they found that the cholesterol absorption efficiency was ranged from 27% to 53% in the subjects but this result was similar to that of previous studies, which was carried out with healthy volunteers and patients with hypercholesterolemia and they have used the same isotope feeding method with deuterium or radioactive isotopes labeled markers. In this study they have performed the experiment on the same subjects second time for measurement of cholesterol absorption and its constant absorption efficiency. They have performed by using a reproducible method and measuring the intraindividual dietary cholesterol absorption. In homozygous subjects for apoE2 or E4, there was no difference found in the absorption efficiency for this study nor there was difference between the ratio of campesterol to cholesterol. The previous observation from Miettinen et al. was same for this study that the ratio of campesterol in plasma is related to the intestinal cholesterol absorption efficiency.
In this study, they did not observed any relationship between percentage of cholesterol absorption and dietary cholesterol and the cholesterol intake was from measured in the range from 62mg/day to 409mg/day. They also did not observed any relationship between cholesterol absorption and the dietary intake of different nutrition and this may be due to their weak point of this experiment of taking small number of subjects in the studies. In this study they did not found any difference in the total cholesterol and bile acid synthesis for individuals with apoε2 and ε4 alleles. They find no difference between different groups in the ratio of lathosterol to cholesterol and total cholesterol synthesis, which conform that there is no difference between the cholesterol synthesis in different groups.
In conclusion we can say that cholesterol absorption efficiency is not related to the apoε until the dietary cholesterol is take in from range 60 to 400mg/day. Evidence for this can be taken from the direct measurement of cholesterol absorption and from the indirect ratio of campesterol to cholesterol in plasma.
Ø Golaz O,Sanchez JC,James RW,Hochstrasser DF. Phenotyping of apolipoprotein E using immobilized pH gradient gels for one-dimensional and two-dimensional separations, Electrophoresis.1995 Jul;16(7):1184-6.
Ø RussellWarnick1Contact Informationand AlanT.Remaley1 Measurement of cholesterol in plasma and other body fluids Current Atherosclerosis Reports Volume 3, Number 5 / September, 2001 Current Medicine Group LLC pages 404-411.
Ø Pavia, Donald L., Gary M. Lampman, George S. Kritz, Randall G. Engel (2006).Introduction to Organic Laboratory Techniques (4th Ed.). Thomson Brooks/Cole. pp.797-817.
Ø Crouse, J. R., M. D. Wilson, and D. S. Weaver. Evaluation of an isotope ratio method for measuring biliary cholesterol secretion. J. Lipid Res. 1983. 2 4 854-860.