Lipids (2012) 47:1063–1071 DOI 10.1007/s11745-012-3708-8
ORIGINAL ARTICLE
Decreased Plasma Cholesterol Concentrations After PUFA-Rich Diets are not Due to Reduced Cholesterol Absorption/Synthesis Vanu R. Ramprasath • Peter J. H. Jones Donna D. Buckley • Laura A. Woollett • James E. Heubi
•
Received: 21 February 2012 / Accepted: 30 July 2012 / Published online: 11 September 2012 Ó AOCS 2012
Abstract Plasma cholesterol concentrations increase with consumption of high saturated fatty acid (SFA) and decrease with high polyunsaturated fatty acid (PUFA) diets, leading to shifts in lipid levels consistent with reduction in heart disease risk. Direct measurements of cholesterol absorption, one of the key regulators of plasma cholesterol levels, have not been performed in humans after consumption of high PUFA diets. Thus, cholesterol absorption and fractional synthesis rates (FSRs) were measured in 16 healthy adults (8 males and 9 females) using a randomized cross-over study with a diet containing high (PUFA/SFA) P/S ratio (2:1) and a low P/S ratio (0.5:1). Cholesterol absorption and fractional cholesterol synthetic rates were measured using stable isotopes after 20 days of dietary intervention. Diet did not affect
V. R. Ramprasath P. J. H. Jones (&) Richardson Centre for Functional Foods and Nutraceuticals, University of Manitoba, 196 Innovation Drive, Winnipeg R3T 2N2, Canada e-mail:
[email protected] D. D. Buckley J. E. Heubi Division of Pediatric Gastroenterology/Hepatology and Nutrition, Children’s Hospital Medical Center, Cincinnati, OH 45229, USA D. D. Buckley J. E. Heubi Department of Pediatrics, Children’s Hospital Medical Center, Cincinnati, OH 45229, USA
cholesterol absorption or synthesis. There was a significant decrease in plasma cholesterol concentrations (P \ 0.02), specifically LDL-cholesterol (P \ 0.02), without a change in HDL-cholesterol or triacylglycerol concentrations. Intraluminal cholesterol solubilization and plasma sterol (cholesterol biosynthetic intermediates and plant sterols) levels were not affected by diet. Thus, consumption of diets with a high P/S ratio reduces plasma total and LDLcholesterol concentrations independent of shifts in cholesterol absorption or synthesis. Keywords Fatty acid Metabolic syndrome Cholesterol Bile Human PUFA SFA Abbreviations PUFA Polyunsaturated fatty acid(s) P/S Ratio between polyunsaturated fatty acid and saturated fatty acid LDL Low density lipoprotein HDL High density lipoprotein SFA Saturated fatty acid(s) CVD Cardiovascular disease RBC Red blood cell CCHMC Cincinnati children’s hospital medical center GCRC General clinical research center FSR Fractional synthetic rate GLC Gas liquid chromatography
D. D. Buckley J. E. Heubi Clinical/Translational Research Center, Children’s Hospital Medical Center, Cincinnati, OH 45229, USA
Introduction
L. A. Woollett Department of Pathology and Laboratory Medicine, University of Cincinnati Medical School, Cincinnati, OH 45237, USA
The type of dietary fat consumed can have a significant impact upon heart disease. Diets high in saturated fatty acids (SFA) often lead to increased plasma cholesterol
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concentrations compared to a diet high in polyunsaturated fatty acids (PUFA) [1–8]. As circulating levels of cholesterol are related to heart disease, including elevated levels of LDL, the ability to reduce plasma cholesterol levels with dietary intervention has been the focus of many studies over the past few decades. Dietary cholesterol can play a role in modulating plasma cholesterol levels. An increase in dietary cholesterol often leads to an increase in plasma cholesterol levels [9, 10]. The impact of dietary cholesterol on plasma cholesterol levels begin with its uptake by enterocytes of the small intestine. Intraluminal cholesterol, dietary as well as biliary cholesterol, is solubilized into micelles containing fatty acids, phospholipids, and monoacylglycerols. The micelles cross the unstirred water layer where they are taken up by protein-mediated processes, i.e. NPC1L1, or passively diffuse into the enterocytes. These chylomicrons reach the systemic circulation where smaller chylomicron remnants are produced which are then taken up by the liver by way of the LDL receptor-related protein (LRP). As most chylomicron remnants are taken up by the liver, a majority of diet-derived cholesterol is taken up by the liver. The diet-derived hepatic cholesterol can then be stored as cholesteryl ester, utilized for cellular maintenance and regulatory processes, or be secreted back into the circulation as VLDL. Circulating VLDL is converted to LDL within the circulation after lipase hydrolysis of core triacylglycerols. The LDL particles are cleared primarily via the LDL receptor [11]. HDL is formed in the circulation as the result of apo AI-mediated efflux of cholesterol from cells with some HDL secreted from the intestine [12, 13]. HDL-cholesterol also can be obtained from VLDL after transfer of ester by way of cholesteryl ester transfer protein (CETP). HDL is taken up by a number of tissues, including the liver and steroidogenic tissues via SR-BI [14]. Various intraluminal factors influence cholesterol absorption, independent of genetic effects on cholesterol transporters. For example, a change in luminal bile acid concentrations and composition can affect cholesterol absorption by altering micellar formation [15–19]. Indeed, we showed that humans given a cholic acid supplement had increased intraluminal bile acid concentrations and consequently more cholesterol carried in non-vesicles, likely micelles [20]. We further showed that cholesterol is absorbed when carried in micelles and not vesicles [21]. A high fat diet also enhances cholesterol absorption, possibly by increasing substrates for micellar formation [22]. Though the relationship between the amount of dietary fat consumed and cholesterol absorption is well known, the effect of dietary fatty acid composition on cholesterol absorption in humans is uncertain. The impact of different fatty acids on cholesterol absorption can be several-fold. First, fatty acids could theoretically affect micellar
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formation. Dietary SFA could remain relatively insoluble, making poor micelles, and/or cholesterol could partition into the undigested lipid. Either effect would result in reduced absorption or reduced plasma lipid levels with SFA, which seems unlikely. In addition, dietary fatty acids can affect expression of genes involved in processes of cholesterol absorption. The PPARs are known mediators of dietary PUFA, as they are activated by PUFA [23]. Recent studies have shown that PPARa can enhance cholesterol absorption by way of NPC1L1 [24]. Though some studies using human subjects have shown a decrease in absorption of cholesterol with diets enriched in PUFA versus SFA [9, 10, 22], the studies have either used indirect measurements of cholesterol absorption (plant sterols), sterol balance measurements, or used nonphysiological diets, i.e. liquid diets or diet that contain extremely large amounts of cholesterol. Thus, the purpose of the current study was to directly measure cholesterol absorption using stable isotopes in humans fed a solid diet. Cholesterol fractional synthetic rates were also measured as these will often be inversely affected by cholesterol absorption. As verification of results, plant sterol and cholesterol biosynthetic intermediate levels were measured as well, as was intraluminal levels of micelles and/or vesicles.
Subjects and Methods Subjects The study was a randomized cross over study and healthy adult males and females of any race (n = 20), ages 18–40 were recruited by advertisement at the University of Cincinnati, College of Medicine and the Cincinnati Children’s Hospital Medical Center (CCHMC). The protocol was approved by the Institutional Review Board of CCHMC as well as the Scientific Advisory Committee of the General Clinical Research Center (GCRC) of CCHMC, Cincinnati, OH, USA (Protocol number #2008-0298). Subjects were screened for any evidence of cardiovascular, pulmonary, renal, gastrointestinal, hepatobiliary diseases or soy allergy and excluded if any conditions were found. Subjects with diabetes mellitus, chronic usage of any medication including oral contraceptives and plasma total and LDL cholesterol exceeding 200 and 120 mg/dl, respectively, were also excluded. Only subjects with Apo AIV 1/1 and Apo E 3/3 genotypes were enrolled in order to reduce the genotypic influence. Apo E and AIV gene expressions could affect cholesterol absorption and account for interindividual variation in total serum cholesterol concentrations [10, 25, 26]. Females were non-pregnant and not planning pregnancy during the course of the study.
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Experimental Design After fulfillment of inclusion/exclusion criteria and a complete verbal explanation of the study, subjects signed the consent form. Subjects provided a 3-day diet diary. Based upon caloric intakes from the diet diary, total calories required to maintain steady body weight were estimated and diet menus were made on a 3 day rotating schedule. During the study, subjects consumed a diet consisting of P/S ratio of 2:1 or a similar diet with a P/S ratio of 0.5:1. The dietary compositions for the two experimental diets are shown in Table 1. Subjects were randomized to diet using a random number table and stratified by age (\30, C30) because of perceived differences in metabolic rates between these age groups. At baseline (day 0) physical examination was performed on the subjects and blood drawn for measuring plasma TAG, total, LDL and HDL cholesterol concentrations. Subjects visited the center daily or every third day to pick up prepared diets which were frozen after preparation. Diets were prepared by the research dietitian on the GCRC at the CCHMC. During each week of the study period, subjects’ body weights were obtained to ensure maintenance of basal weight. Subjects were advised not to eat anything other Table 1 Composition of the experimental diets Composition Proteins (g/100 kcal)
Low P/S
High P/S
5.04
4.94
Carbohydrates (g/100 kcal)
12.56
12.59
Fats (g/100 kcal) Cholesterol (mg/100 kcal)
3.28 15.05
3.32 12.05
6:0
0.07
0.10
8:0
0.11
0.10
10:0
0.21
0.15
12:0
0.21
0.30
than the foods provided. Subjects were also instructed to follow the dietary instructions such as eating all the meals on time and to maintain their physical activity to be same throughout the study. No alcohol was permitted during the whole study period. During and at the end (day 14 and 19, respectively) of each phase, fasting blood samples were collected for analysis and urine for pregnancy testing for females. After 2 weeks on the diet (day 14), fasting blood samples were collected from study subjects for analysis. Plasma was separated for measuring TAG, as well as total, LDL and HDL cholesterol concentration measurements. 2 days later (day 16) subjects were provided with an intravenous injection of 15 mg [25,26,26,26,27,27,27-D7] cholesterol and a 75-mg oral dose of [3,4-13C] cholesterol for cholesterol absorption analysis according to the method of Bosner et al. [27], as originally proposed by Zilversmit [28] and as described previously [29]. The ratio of ingested 3,4-13C cholesterol to injected 25,26,26,26,27,27,27-D7 cholesterol enrichment in red blood cells (RBC) cholesterol after 48, and 72 h was taken as an indicator of the cholesterol fractional absorption rate. Cholesterol synthesis rates were also measured using the deuterium incorporation approach. On day 19, after an overnight fast, baseline blood was drawn for RBC cholesterol isotope measurement. Subjects were given oral deuterated water, and the next day (day 20) blood was obtained at the same time as the isotope administration on day 19. On completion of first dietary period, subjects participated in the alternative arm of the study after a washout period of at least 4 weeks. Thereafter, the same sequence of events was performed as described for the first phase of the study.
Fatty acids (g/100 g of fat)
14:0
1.82
0.94
16:0
22.15
19.77
18:0
10.43
8.48
20:0
0.04
0.05
16:1
2.85
1.82
18:1
32.94
27.76
20:1
0.14
0.20
18:2
23.18
34.27
18:3
2.88
3.50
SFA (g/100 kcal) PUFA (g/100 kcal)
1.09 0.56
0.55 1.14
MUFA (g/100 kcal)
1.37
1.29
P/S polyunsaturated/saturated fatty acids, SFA saturated fatty acids, PUFA polyunsaturated fatty acids, MUFA monounsaturated fatty acids
Plasma Lipid Concentrations Blood samples after collection were centrifuged at 1,500 rpm for 15 min to separate RBC and plasma. Separated aliquots were immediately stored at -80 °C until analysis. Plasma total and HDL cholesterol and TAG concentrations were measured enzymatically by Quest Diagnostics Nichols Institute, a certified Center for Disease Control (CDC) Lipid Research Clinic. Desmosterol and lathosterol levels were measured in plasma as markers for cholesterol synthesis and campesterol and b-sitosterol levels were measured as cholesterol absorption markers [30]. Sterol concentrations were determined by gas liquid chromatography (GLC), as described by Ntanios and Jones [31], using 5a-cholestane (Sigma-Aldrich Canada Ltd, Oakville, ON, Canada) as an internal standard. Briefly, after saponification, non-saponified materials were extracted and derivatized with TMSi reagent [pyridine-hexamethyl disilazantrimethyl chlorosilane (9:3:1,
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vol:vol)] [32]. Sterols were identified using known standards (Sigma-Aldrich Canada Ltd, Oakville, ON, Canada).
Cholesterol Absorption Free cholesterol was extracted from RBC according to established methods [33]. 13C cholesterol enrichments in RBC lipid extracts were determined using on-line gas chromatography/combustion/isotope ratio mass spectrometry approach (Agilent 6890N chromatograph interfaced with a Finnigan Delta V Pulse isotope ratio mass spectrometer (Bremen, Germany)). Isotope abundance, expressed in delta (d) per mil (%), was calculated using CO2 as a reference gas and further corrected against the international reference standard, Pee Dee Belemnite limestone. The measurement of free-cholesterol deuterium enrichment was performed using online gas chromatography/pyrolysis/isotope ratio mass spectrometry and expressed relative to standard mean ocean water and a series of standards of known enrichment. Isotope abundance, expressed in d %, was calculated using H2 as reference gas. The average 13C and D7 enrichments of 48 and 72 h RBC free cholesterol relative to baseline samples were used to calculate the cholesterol absorption coefficient using the ratio of orally ingested 13C cholesterol to intravenously administered D7 cholesterol as described by Bosner et al. [27] and by us [29]. Cholesterol Fractional Synthetic Rate Cholesterol synthesis rates were assessed after 24 h of deuterium water administration using the deuterium incorporation approach [34] as described before [29]. This method measures cholesterol synthesis as the rate of deuterium incorporation from body water into RBC membrane free cholesterol over a 24 h period. Deuterium enrichment was measured in both RBC free cholesterol and plasma water as described above. Enrichments were expressed relative to standard mean ocean water using a calibration curve of working standards. FSR is taken to represent RBC free-cholesterol deuterium enrichment values relative to the corresponding mean plasma water sample enrichment after correcting for the free-cholesterol pool. FSR represents that fraction of the cholesterol pool that is synthesized in 24 h and is calculated as per the equation [34].
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the tube tip placed at the ligament of Trietz, and 12–20 ml of duodenal drainage was collected by siphonage for 15 min (-15 to 0 time). Subjects then ingested a meal consisting of olive oil, egg and egg white, sucrose, vanilla extract and 0.15 M NaCl in 240 ml water [29]. Duodenal drainage was collected in 15 min intervals for 90 min and then one 30 min interval. Anti-microbial and anti-bacterial compounds were added as samples were collected. After completion of collection periods, 0.5 ml aliquots were saved to measure lipids in luminal contents and remaining 12–15 ml separated into the subphase. The subphase was separated into vesicles and non-vesicles by size-exclusion column chromatography as described [35]. Cholesterol was measured directly in the total and subphase intraluminal contents by GLC; the ratio of subphase to total cholesterol was considered solubilized cholesterol. Cholesterol was also measured in the fractions from the size exclusion columns mostly by GLC. Vesicular cholesterol was that which came off the column in the void volume and nonvesicular cholesterol, including micellar cholesterol, was that which was eluted from the column after the initial vesicular fraction. Apolipoprotein Genotypes DNA from peripheral blood was isolated according to instructions provided in the High Pure PCR Template Preparation Kit (Boehringer Mannheim, Indianapolis, IN, USA). ApoE and ApoA-IV genotypes were determined as described by earlier [36, 37]. Statistical Analysis Results were expressed as means ± SEM obtained from ANOVA using the root mean square error to estimate the pooled standard error. End point variables were tested for statistical significance by student’s t test and repeated measures ANOVA with p \ 0.05. The correlations among plasma lipid profile, cholesterol absorption and synthesis, cholesterol precursors, sterols and their ratios were analyzed using Pearson correlations. All statistical measures were analyzed using Statistical Package for the Social Sciences (SPSS) version 10.0.
Results Intraluminal Cholesterol Solubilization After blood collection on day 14, duodenal aspirates were collected and treated as described [29]. Topical anesthesia was applied to the nose and throat, and sedation was used with intravenous midazolam if subjects wished. A nasoduodenal tube was placed with fluoroscopic guidance with
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Seventeen subjects (8 males and 9 females) with an average of 26.5 ± 3.6 years of age, 70.9 ± 16.6 kg and BMI of 24.3 ± 3.3 kg/m2 (Table 2) started the study of which 16 completed both phases of study. Fifteen Caucasians and one African subject participated. One subject withdrew from the study after completing first phase which was a
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Cholesterol absorption
Table 2 Baseline characteristics of subjects Subjects (n = 17)
Age (years)
26.47 ± 3.57
Body weight (kg)
70.89 ± 16.65
Height (cm)
169.63 ± 13.01
Body mass index (kg/m2)
24.3 ± 3.27
Total cholesterol (mg/dl)
163.4 ± 26.5
LDL (mg/dl)
95.1 ± 21.9
HDL (mg/dl)
51.0 ± 19.47
Triglycerides (mg/dl)
89.0 ± 34.6
high ratio P/S diet due to personal reasons and not related to the study. No adverse events were recorded during the study due to the diet. No significant changes were observed in body weights when comparing both between the phases and between the baseline and end points of each phase. Plasma total cholesterol (P = 0.015) and LDL-cholesterol (P = 0.014) concentrations were reduced as early as 14 days of consumption of a diet with a high P/S ratio (Table 3). By day 19 of the study, a further reduction in total (P = 0.011) and LDL-cholesterol concentrations (P = 0.003) occurred such that total cholesterol was reduced &8 % and LDL-cholesterol was reduced &12 % (Table 3). No impact of diet on HDL-cholesterol or TAG concentrations was observed at either time point after diet consumption. No significant difference was found in fractional cholesterol absorption determined by dual stable isotope kinetics measurement (Fig. 1). Cholesterol absorption markers including b-sitosterol and campesterol showed no significant changes in their concentrations as well their ratios with cholesterol in plasma between high and low P/S ratio diet phases (Table 4). In parallel with results, there was no difference in the solubilized cholesterol in the
Table 3 Lipid profile of subjects during and after consuming a high or low P/S ratio diet Variables
Total cholesterol (mg/dl)
LDL (mg/dl)
HDL (mg/dl)
TG (mg/dl)
High P/S (day 14)a
147.2 ± 7.5*
83.4 ± 6.3*
47.7 ± 4.0
81.1 ± 6.0
Low P/S (day 14)a
158.6 ± 7.6
94.4 ± 6.4
47.1 ± 3.4
87.0 ± 6.9
High P/S (day 19)b
144.1 ± 9.2*
79.9 ± 7.1*
49.3 ± 4.5
74.7 ± 6.8
Low P/S (day 19)b
155.8 ± 9.8
91.8 ± 7.4
49.5 ± 4.5
74.6 ± 8.5
Values are expressed as means ± SEM * Significant at P \ 0.05 compared with low P/S ratio diet at same time points a n = 16, b n = 13
0.8
Pool/Pool
Variables
1
0.6 0.4 0.2 0 High P/S
Low P/S
Fig. 1 Cholesterol absorption for each subject fed a high or low P/S ratio diet (n = 13)
Table 4 Plasma sterols concentration in plasma and their ratios with cholesterol and cholesterol precursors in subjects after consuming a high or low P/S ratio diet Variables
Low P/S
High P/S
Campesterol (lmol/l)
11.3 ± 1.0
11.6 ± 0.7
b-Sitosterol (lmol/l)
6.7 ± 0.8
7.0 ± 0.7
18.0 ± 1.5 2.4 ± 0.3
18.6 ± 1.2 2.9 ± 0.2
Campesterol ? b-sitosterol (lmol/l) Campesterol:cholesterol (lmol/mmol) b-Sitosterol:cholesterol (lmol/mmol)
1.4 ± 0.2
1.8 ± 0.2
(Campesterol ? b-sitosterol):cholesterol (lmol/lmol)
3.8 ± 0.4
4.6 ± 0.3
Lathosterol:campesterol (lmol/lmol)
1.0 ± 0.2
1.0 ± 0.2
Lathosterol:b-sitosterol (lmol/lmol)
1.7 ± 0.4
1.6 ± 0.4
Values are expressed as means ± SEM (n = 10)
lumen of the subjects containing either diet (Fig. 2a). As might be expected, the percentage of non-vesicular cholesterol also remained similar between all subjects (Fig. 2b). No apparent change was seen in cholesterol absorption, or fractional synthetic rate of cholesterol with different dietary fatty acid composition (Fig. 3). Indirect measures of cholesterol synthesis, plasma cholesterol precursor levels, i.e. desmosterol and lathosterol, failed to significantly differ between groups (Table 5).
Discussion Our results confirm that diets consisting of greater P/S ratios had reduced plasma cholesterol concentrations, specifically LDL-cholesterol [3, 5, 38–40]. Importantly, our results specifically show that the decrease in LDLcholesterol levels was not due to an effect of dietary fatty acid composition on cholesterol absorption or synthetic rate. Support for these results was confirmed by indirect
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A
0.14
LOW PUFA HIGH PUFA
100 80 60
Cholesterol FSR
0.12
Pool/day
SOLUBILIZED CHOLESTEROL (%)
120
0.1 0.08 0.06 0.04
40
0.02
20
0 High P/S
0
-20
0
20
40
60
80
100
120
TIME (min post-meal)
B
LOW PUFA HIGH PUFA
NON-VESICULAR CHOLESTEROL (%)
80
Low P/S
Fig. 3 Cholesterol fractional synthesis rate for each subject fed a high or low P/S ratio diet (n = 13) Table 5 Plasma cholesterol precursor concentration in plasma and their ratios with cholesterol in subjects after consuming a high or low P/S ratio diet Variables
Low P/S
High P/S
60
40
Desmosterol (lmol/l)
12.0 ± 0.9
11.6 ± 1.1
Lathosterol (lmol/l)
11.6 ± 1.5
11.14 ± 1.6
Desmosterol:cholesterol (lmol/mmol)
2.5 ± 0.3
2.9 ± 0.2
Lathosterol:cholesterol (lmol/mmol)
2.5 ± 0.4
2.7 ± 0.3
20
Values are expressed as means ± SEM (n = 10) 0 -15 to 0
15 to 30
45 to 60
TIMES POST-MEAL (min) Fig. 2 The amount of cholesterol solubilized in the aqueous fraction (subphase) of the luminal samples of subjects fed low or high PUFA diets. After an overnight fast, a naso-duodenal tube was placed at the ligament of Trietz and 15–20 ml of luminal sample collected. Subjects consumed a liquid meal as described in the study design section of the Research Plan, and samples collected from the lumen every 15 min for 2 h and then for 30 min. Samples were spun to separate the subphase from total contents. The amount of cholesterol in the total and subphase was measured by GLC and amount solubilized shown (a). The percentages of total cholesterol present as non-vesicular cholesterol in the subphase at -15 to 0, 15 to 30, and 45 to 60 min post-meal are shown (b). Data are presented as means ± SEM (n = 16)
measurements of cholesterol trafficking. Specifically, the extent of non-vesicular cholesterol, likely present as micelles, was the same in lumens of subjects on either diet. As cholesterol needs to be packaged into micelles prior to absorption [21], it is not surprising that cholesterol absorption was not different. Likewise, circulating plant sterol concentrations were similar, an indication of similar cholesterol absorption [30]. For cholesterol synthesis, cholesterol biosynthetic precursors in the circulation were similar as well [30]. Until now, the relationship between dietary fat composition and cholesterol absorption in humans has remained unclear since methods used included different indirect
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absorption measurements, variably controlled subject populations, and variations in dietary cholesterol. Unlike our study, in which dietary cholesterol was rigorously controlled, subjects were normolipemic, subjects from specific apo E genotypes, and cholesterol absorption measured by the dual isotope method; most previous studies were much less carefully controlled using state-ofthe-art methods at the time of publication. McNamara et al. [9] evaluated the effect of a dietary cholesterol challenge within diets containing either saturated or polyunsaturated fat. Fractional cholesterol absorption was not affected by the P/S ratio, regardless of amount of cholesterol fed. Grundy and Ahrens directly compared cholesterol absorption in subjects given SFA versus high P/S diets containing relatively high levels of dietary cholesterol and observed no differences in fractional cholesterol absorption and marked variability [41]. Using a cross over design, Wood et al. [22] studied the effect of liquid formula containing dietary fat as predominantly saturated or unsaturated fat plus high levels of cholesterol in five normolipidemic adults and found decreased cholesterol absorption when subjects received diets containing predominantly unsaturated fat. Finally, Miettinen et al. discovered that cholesterol absorption decreased when changing from a diet relatively high in cholesterol and fat (38 % calories) with mostly saturated fat (P/S = 0.28) to one with half as much cholesterol and less fat (24 % calories) with a higher P/S
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ratio (0.72). Even though there was less amount of fat and cholesterol in the diet, impact of changes with dietary fat composition on cholesterol was very large [10]. A number of additional studies have been completed in which sterol synthesis was measured. As reduced cholesterol absorption will lead to increased sterol synthesis, these studies could also help delineate the effect of dietary fatty acid on cholesterol absorption. Interestingly, a number of studies found that the fractional synthetic rate for cholesterol was higher in subjects fed PUFA-containing diets compared to baseline diets or diets containing high SFA [40, 42–45]. If PUFA only affected absorption, these studies would suggest that PUFA affected cholesterol absorption in subjects of these other studies. However, other variables exist that can lead to a separation of the two processes. For example, different fatty acids can have direct impact upon metabolism. Likewise, some of these studies used hypercholesterolemic or obese subjects. In support of our absorption data, we detected no differences in cholesterol synthesis by stable isotope or by cholesterol biosynthetic intermediate concentrations. Cholesterol absorption measurements have also been carried out in various animal models. Studies in rats and rabbits have shown that dietary PUFA either increased or decreased cholesterol absorption [46–48]. Mott et al. showed cholesterol absorption to be similar in baboons fed diets containing either saturated or unsaturated fat independent of cholesterol content [49, 50], whereas Tanaka and Portman [51] showed that PUFA versus SFA-enriched diets lead to increased cholesterol absorption in squirrel monkeys. Part of the differences in the various models are likely due to variations in cholesterol metabolism [50], making the current studies important and relevant as they were completed in humans. Even though there was no effect on cholesterol absorption in the current studies, plasma cholesterol concentrations were still reduced in subjects consuming a greater P/S ratio. Over the years, several different processes have been shown to be affected by dietary PUFA. Daumerie et al. [52] demonstrated a reduction in hepatic LDL receptor activity and elevated LDL cholesterol production rate in hamsters fed with a SFA-enriched diet compared to one enriched in PUFA or MUFA, ultimately leading to increased plasma LDL-cholesterol concentrations [52–54]. Perhaps SFA have a greater effect on increasing plasma cholesterol versus the impact of PUFA on decreasing plasma cholesterol levels, depending on the baseline or comparison. This is actually supported by equations devised by Keys et al. [55] and Hegsted et al. [2] and more recently by Etherton and colleagues [56] that gives a relative change in plasma cholesterol based on the nature of the fat fed. Other effects of lipids on metabolism might also occur. In addition, high PUFA containing diets
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lead to reduced activities of lipogenic enzymes such as phosphatidase, phosphohydrolase and diacylglycerol acyltransferase compared with low PUFA containing olive oil and thereby decrease serum lipid concentrations [57]. Consumption of a diet with high P/S ratio also increases postprandial fat oxidation [42, 58]. Thus, some of these non-absorptive effects of PUFA must have played a role in the reduction of plasma cholesterol when higher P/S diets are fed to normocholesterolemic subjects. In conclusion, consumption of a high P/S ratio diet did not affect cholesterol absorption or synthesis in normolipidemic subjects, despite a decrease in total and LDL cholesterol concentrations. Results indicate that high P/S diets alter lipids through mechanisms other than cholesterol absorption and synthesis in normolipidemic subjects. Acknowledgments We thank YenMing Chan, Richardson Centre for Functional Foods and Nutraceuticals, and Dr. Scot Harding, adjunct professor, Department of Human Nutritional Sciences, University of Manitoba for their help with the analytical part of the study. We also thank Suzanne Summer and the staff of the Body Composition Core of the General Clinical Research Center for their help with dietary management for the study. We were also supported by the National Institute of Health RR 08084 and DK 068463. Conflict of interest current study.
No authors have any conflict of interest with the
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