In this study we aimed to establish the reproducibility of the serum-lipid response to coffee oil. We found that the response of total serum cholesterol and LDL to coffee oil was poorly reproducible, whereas the responses of HDL and triglycerides to coffee oil proved to be highly reproducible. A high correlation between two intra-individual responses means that the within-subject variability is relatively low. The between-subject variability of HDL and triglycerides is relatively large. The within-subject reproducibility of the responses of HDL and triglycerides to coffee oil in combination with their large between-subject variability indicates that it is more promising to investigate genetic variation that determines the response of HDL and triglycerides to coffee oil than to investigate the responses of total and LDL cholesterol.
The triglyceride response is relatively large compared to the response of total cholesterol. In our study cholesterol increased on average by 21 % and triglycerides by 62%. It has been shown that cafestol increases plasma triglycerides by an increasing production of a fraction of very low density lipoproteins: VLDL
1. The subsequent rise in LDL cholesterol might be caused by enrichement of VLDL
2 particles with cholesteryl esters [
14].
Group vs. individual response of serum lipids
The average response to the coffee-oil treatment was similar to that in previous studies at our department [
11]. We expected an increase in total serum cholesterol of 1.0 mmol/l and an increase of serum triglycerides of 0.65 mmol/l, which is close to the observed values (Table
2). The coffee oil did not affect the average HDL concentration. Other studies also found either no effect on HDL or a slight decrease in HDL concentrations [
11].
The assessment of the individual response of serum lipids is hampered by day-to-day fluctuations in serum lipid levels [
3‐
5]. In this experiment we reduced the effect of these fluctuations by using the mean of four separate measurements [
15,
16].
Analyses of the between and within-person standard deviations (SD's) of the responses of serum lipids to coffee oil confirm that total cholesterol and LDL responses are poorly reproducible. The SD's of the responses of total cholesterol and LDL had a within-person component that was clearly larger than the between-person component. For the total cholesterol response to coffee oil we found a total SD of 0.49 mmol/l, an SD
within of 0.44 mmol/l, and an SD
between of 0.22 mmol/l. This is at variance with the results of Katan
et al who observed an SD of 0.33 mmol/l, an SD
within of 0.16 mmol/l, and an SD
between of 0.29 mmol/l for total cholesterol [
2]. This would indicate a better reproducibility than observed in our study. However, Katan
et al selected putative hyperresponders and hyporesponders which will lead to overestimation of the reproducibility. In our study the response of triglycerides shows a large SD between individuals and a quite small SD within persons. This confirms that the response varies between individuals and that the response is reproducible within persons.
We did not separate laboratory variation in our model for calculation of SD's. We estimated this variation by measuring serum lipids in 32 duplo samples. Coefficients of variation were 1.6% for total cholesterol, 1.8% for HDL, and 2.3% for triglycerides. This means that the laboratory variation is so small that it can be omitted from the model without affecting the calculated values of SDwithin and SDbetween.
Study limitations
Although cafestol is a potent cholesterol-raising food component, it is not certain that it can be used for the study of variation in genes regulating the serum-lipid response to other foods. It is possible that cafestol regulates different genes than other food components such as dietary cholesterol or saturated fats do. Furthermore, we did not use pure cafestol in this study, but coffee oil, which contains many more components, such as triglycerides, free fatty acids, and sterols. However, coffee-oil stripped of diterpenes has no effect on serum lipids [
9]. Therefore, genes that cause a rise in serum cholesterol are probably affected by diterpenes.
Another limitation of the study is that the subjects were free living and did not receive a controlled diet. Therefore, the response of serum lipids to the coffee oil could be changed by other factors. We instructed the subjects to maintain dietary habits, smoking habits, and physical activity. Changes were recorded in a diary together with use of medication and illness. According to the diaries, subjects maintained their habitual lifestyle. Furthermore, coffee oil has such a large effect on serum-lipid levels that small effects of other factors are not of great concern.
A fourth limitation is the large drop out due to elevations of liver enzymes. There was, however, no correlation between the rise in serum-lipid levels and the rise in liver enzymes (data not shown). Therefore, there is no reason to assume that the reproducibility of the serum lipid response to coffee oil in subjects who showed a considerable increase of liver enzymes differs from the serum lipid response in subjects who did not show a large increase of liver enzymes.
On basis of this study it can not be concluded with certainty that a response to coffee oil that differs between individuals but is reproducible within individuals is determined by genetic variation.
Liver enzymes
Levels of the liver enzymes ALAT and ASAT rose after administrating coffee oil, as was expected from previous studies [
9,
11,
12,
17]. The rises in ALAT and ASAT indicate that coffee oil can cause acute injury to hepatocytes [
18,
19]. Alcohol, being hepatotoxic, might be an important cofactor in this effect [
20]. In this study no association between use of alcohol and drop out due to elevation of liver enzymes was observed. Levels of ALAT rose more than ASAT levels did. This could mean that the membranes of hepatocytes were damaged [
18,
20].
Genetic factors underlying the serum lipid response to cafestol
Given the ratio of between and within person variability of the responses of HDL and triglycerides to coffee oil, research into genetic determinants of the response seems to be feasible.
Other studies have described polymorphisms in genes that have a small effect on the total-cholesterol response to cafestol and dietary fat [
6‐
8]. An example is the cholesteryl ester transfer protein (CETP). CETP is a protein that mediates the transfer of cholesteryl esters from HDL to LDL and VLDL. Cafestol, like dietary cholesterol and fat, might increase the transfer of cholesteryl esters by increasing the activity of CETP. Weggemans et al. showed that humans with the CETP Taqlb-1/2 allele have a smaller response of LDL to cafestol, or dietary fat and cholesterol [
21]. However, it remains to be established whether CETP has a role in the cholesterol-raising effect of cafestol and whether a polymorphism in the gene accounts for some of the variation in response between individuals.
Other candidate genes are genes encoding for proteins in the bile acid metabolism. Cholesterol is converted to bile acids in the liver. There are two pathways involved: the neutral and the acidic pathway. The rate-limiting enzyme in the neutral pathway is 7α-hydroxylase, which is regulated by bile acids through a negative feedback mechanism [
22]. Chenodeoxycholic acid (CDCA), a primary bile acid, suppresses 7α-hydroxylase activity by binding directly to the farnesoid X receptor [
23,
24]. It has been shown in mice that cafestol inhibits bile acid synthesis, which could cause the rise in serum cholesterol [
25]. If functional polymorphisms are present in the genes involved in bile acid metabolism, these could be responsible for the variation in the conversion of cholesterol to bile acids. Therefore, it is interesting to study whether cafestol regulates genes involved in bile-acid metabolism.
Polymorphisms in the genes of the pathways mentioned above could account for the variation in response to cafestol between individuals. There are more possible candidates, such as sterol regulatory element binding proteins, microsomal triglyceride transfer protein, lecithin:cholesterol acyltransferase, lipoprotein lipase, and hepatic lipase.
In this study the responses of HDL and triglycerides to coffee oil showed a sufficient reproducibility. Therefore, our best option is to focus on these serum lipids and characterize an individual's response to coffee oil by the HDL and triglyceride response in future research. The variation between persons in the response of HDL and triglycerides combined with the consistency within persons does not guarantee that a large genetic effect is present. It means that environmental determinants of the response were stable within individuals during the time span of the study, whereas there were differences between individuals in other environmental determinants and/or genetic determinants. The question remains whether possible genetic effects are large enough to be detected and whether variation in these genes is sufficiently prevalent in the population.
Clarification of the mechanism by which cafestol increases serum lipids might provide leads for dietary and pharmacotherapeutical ways to lower serum cholesterol. The 'cafestol model' also could be a trial case for evaluating the possibility of personalized diets. If it is possible to predict people's serum-lipid responses to cafestol on basis of their genetic make-up and to make dietary recommendations based on this genetic information, this could also be applicable in other situations.