Evidence suggests that dietary calcium intake may be inversely related to body weight. One explanatory mechanism is that dietary calcium increases fecal fat excretion, due to either calcium soap formation and/or binding of bile acids (BAs) in the intestine.
To examine the effect of calcium from low-fat dairy products on fecal fat excretion.
A randomized crossover study with 11 subjects, comparing two 7-d diets: one high in calcium from low-fat dairy products (high-Ca; 2300 mg Ca per d) and one low in calcium (low-Ca; 700 mg Ca per d).
All feces were collected during the last 5 days of each diet period and analyzed for fat, energy and calcium content and fatty acid (FA) and BA composition.
Dairy calcium significantly increased the total fecal fat excretion from 5.4±0.5 g d−1 on the low-Ca diet to 11.5±1.4 g d−1 on the high-Ca diet (P<0.001). The fecal energy excretion increased almost correspondingly. Saturated, monounsaturated and polyunsaturated FAs were all excreted in larger amounts on the high-Ca diet (P<0.001 for all), with the effect of calcium being greater for monounsaturated than for saturated FAs. The fecal excretion of BAs was unaffected of calcium intakes.
Increasing the intake of calcium from low-fat dairy products by 1600 mg d−1 for 7 days doubled total fecal fat excretion, but did not affect the excretion of BAs. The results may partially explain why a high-calcium diet can produce weight loss.
An inverse association between calcium intake and body weight in humans was reported for the first time in the mid-1980s, based on data from the first National Health and Nutrition Examination Survey.1 More than a decade later, an increased fat loss of almost 5 kg, due to an augmented calcium intake from dairy products, was observed in an antihypertension trial.2 Since then, increasing evidence that dairy calcium reduces body weight has emerged from both observational and intervention studies.3, 4, 5, 6, 7 However, not all investigations have confirmed these findings.8, 9, 10, 11, 12, 13, 14
The mechanism responsible for the effect of increased calcium intake on energy balance is not clear, but a number of different mechanisms have been suggested. One possible explanation, proposed by Zemel et al.2, is that serum calcium plays a regulatory role in lipid metabolism by influencing intracellular calcium levels through hormonal regulation. According to this hypothesis, an increase in dietary calcium would result in increased lipolysis and decreased lipogenesis, thereby stimulating body fat loss. An alternative, or additional, possibility is that calcium interferes with fat absorption in the intestine by forming insoluble calcium soaps with fatty acids (FAs) or by forming precipitates with phosphate and bile acids (BAs), resulting in decreases in the digestible energy of the diet.15, 16, 17, 18
A decrease in fat absorption, inferred from increases in fecal fat excretion, has been observed in several studies. Previous research from our group showed a 2.5-fold increase in fecal excretion of fat (from 6 to 14.2 g d−1) when the dietary intake of calcium from low-fat dairy products was increased by 1300 mg d−1.19
Others have observed quantitatively smaller effects (up to 4.0 g d−1) on fecal fat loss when augmenting dietary calcium from different sources.20, 21, 22, 23 Recently, Boon et al.24 observed a tendency toward a higher fat excretion on diets with high dairy calcium content compared with a diet with low calcium content, but the difference failed to reach statistical significance.
It has been suggested that, especially, the absorption of saturated fat is affected by dietary calcium.20, 25 This is of particular interest because saturated fat has been associated with adverse effects on cardiovascular health.26
The primary aim of this study was to verify the results of our previous investigation and thereby to examine whether a high calcium intake from low-fat dairy products increases fecal fat and energy loss in diets with normal protein content. Additional aims were to examine whether dietary calcium affects the fecal loss of all major FAs and to examine whether BA binding in the gut can be an explanatory mechanism behind the effect of dairy calcium on fecal fat loss.
Subjects and methods
Eleven subjects (five men and six women) were recruited through advertisements posted on university web pages and in local shops and higher educational institutions. Subject inclusion criteria were healthy, moderately overweight (body mass index (BMI) between 25 and 31 kg/m2) and aged 18–50 years, extremes included. The exclusion criteria were hypertension (>140/90 mm Hg), lactose intolerance, infections or metabolic diseases. The subjects were not allowed to use dietary supplements during the study and 1 month before commencement. Elite athletes were excluded, and female subjects were not to be pregnant or lactating.
We certify that all applicable institutional and governmental regulations concerning the ethical use of human volunteers were followed during this research. The subjects were given both verbal and written information, whereupon all gave written consent. The study was carried out at the Department of Human Nutrition, Faculty of Life Sciences, University of Copenhagen, Frederiksberg, Denmark, and was approved by the Municipal Ethical Committee of Copenhagen and Frederiksberg in accordance with the Helsinki-II declaration (KF 01-144/02). Subjects received 3000 Danish Crowns (∼500 US$) as compensation on completion of all the tests.
The study had a randomized crossover design, where two different isocaloric diets were examined. The subjects were randomly assigned to the sequence of the diets. There was a washout period of 1 week between the two 7-d dietary intervention periods.
All feces were collected in pre-weighed plastic containers on the last 5 days of each diet period. Transit time was measured by using non-absorbable radio-opaque transit markers. With breakfast on days 1–5 of each diet period, the subjects ingested capsules containing transit markers (20 markers per d), differing in size and shape for each day. Transit time was determined as described elsewhere.27 As the scope of this study was to compare the effects of a high- versus a low-dairy calcium diet, the baseline fecal fat excretion was not assessed. Body weight and blood pressure were measured at screening and on the mornings of days 1 and 8, and complete 24-h urine collections were carried out on day 7 of each diet period. During the 24-h urine collections, the subjects ingested a tablet of 80 mg para-amino-benzoe acid three times a day (a total of 240 mg d−1). A para-amino-benzoe acid recovery of >85% was considered complete.28
All food was provided by the Department of Human Nutrition, in daily portions matching the individual subject's energy requirement. Most of the food was pre-portioned. The dinner meals were ready-prepared and only had to be heated up. Subjects weighed some of the food items themselves, in accordance with the diet plan, on digital kitchen scales issued by the Department. The subjects were instructed to follow the diet plan very strictly and report any deviation from the diet plan. They were allowed to consume water, tea, coffee and diet soft drinks ad libitum, of which they were told to keep record.
The subjects were instructed to maintain their habitual activity level throughout the study. During their first diet period, they were taught to keep an exercise diary, recording the type and duration of physical activity for each day. The subjects were told to follow the same activity pattern in the second diet period. The subjects' habitual calcium intake was assessed at screening by using a self-administered quantitative food frequency questionnaire. The food frequency questionnaire has been validated against food records, and its reproducibility and validity have previously been described in detail.29
Two isocaloric study diets were examined, one diet high in calcium (high-Ca), from low-fat dairy products, and one low in calcium (low-Ca). The composition of the diets, as normalized per MJ, is presented in Table 1.
The macronutrient composition, including the fat composition with respect to saturated FAs (SFAs), monounsaturated FAs (MUFAs) and polyunsaturated FAs (PUFAs), was designed to be similar in both diets as was the content of vitamin D and dietary fiber. The content of macro- and micronutrients in each diet was estimated using the Dankost 3000 dietary assessment software (Danish Catering Center, Herlev, Denmark).30 The calcium, fat, FAs and energy content were analyzed chemically as described below.
The test diets were prepared at the department from normal Danish food items. The main source of calcium in the two diets was low-fat dairy products. The main sources of fat in both diets were dairy (butter) fat, cocoa butter and rapeseed oil. The main protein source in the low-Ca diet was pork meat, whereas most of the protein in the high-Ca diet was of dairy origin. Each diet consisted of one breakfast dish, one lunch dish, two snacks and two different dinner meals, which were served on alternate days. Portions were matched to each subject's individual energy requirement adjusted to the nearest 1 MJ. The energy requirement of each subject was calculated as basal metabolic rate (BMR) × physical activity level (PAL). The BMR was estimated using the following equations from the Nordic Nutrition Recommendations.31
An overall PAL value was calculated as ‘basis PAL+0.025 × active hours’, where a basis PAL value of either 1.4 or 1.7 was applied according to general lifestyle.31 The average number of active hours daily was assessed using a questionnaire.32
Calcium in feces and urine, and nitrogen in urine were used as indirect measures of compliance with the diet plan. The net retention of calcium (the difference between the dietary intake of calcium and that which is excreted in the feces, urine and sweat) for adults is on an average 10–30 mg d−1.33 As normal healthy subjects normally only loose small amounts of calcium through sweat, shed skin, hair and nails (∼60 mg d−1), the total excretion of calcium in urine and feces should approximately balance out the calcium intake.34 Excretion of nitrogen in urine is a marker of protein intake. Under normal circumstances, ∼81% of ingested protein is excreted in urine as nitrogen.35
Anthropometrics and blood pressure
Body weight was measured in kilograms with two decimals by a Lindeltronic 8000 scale (Sweden). Height was assessed twice at baseline to the nearest half-centimeter using a Seca stadiometer (Hultafors, Sweden) and the average of the two measurements was recorded.
Blood pressure measurements were performed in the seated position after 10 min of rest, with an automatically inflated cuff (UA-787; A & D Co Ltd, Saitama, Japan). At baseline, blood pressure was measured in both arms, and all subsequent measurements were performed on the arm showing the highest systolic blood pressure. A minimum of three measurements was performed at each visit. If the last two measurements differed with 5 mm Hg or less, their average value was recorded. If not, additional measurements were performed until two subsequent measurements differed less than 5 mm Hg.
Before analysis, the fecal samples were freeze-dried and homogenized. For each subject, samples from the same diet period were pooled. Fecal energy was obtained using a bomb calorimeter (Ika-calorimeter system C4000; Heitersheim, Germany). Calcium in feces was measured by atomic absorption spectrophotometry using a PYE UNICAM SP9 atomic absorption spectrophotometer (Philips Electron Optics, Mahwah, NJ, USA). Before analysis, the samples were destroyed by dryashing at 450 °C for 5 h and the ashes were dissolved in HNO3 (21.7%). Before the calcium concentration was measured, the solution was diluted with a lanthanum chloride solution.
Total fecal fat was analyzed after the method of Bligh and Dyer,36 with modifications. Before fat extraction, the fecal samples were acid hydrolyzed with 3 N HCl at 80 °C for 1 h. During extraction, a known quantity of 17:0 (Sigma H-4515) was added to all samples as an internal standard for the FA analysis.
The fecal FA composition was determined as described below. The extracted fat was saponified with 0.8 ml methanolic sodium hydroxide (0.5 M) by heating at 100 °C for 1 h. The FAs were hydrolyzed by the addition of 1 ml of boron triflouride/methanol complex (20%) and methylated by heating at 100 °C for 45 min. The methyl esters were extracted by 2 ml heptane mixed with 4 ml of saturated sodium chloride solution. Finally, the heptane fase was transferred to vials. The FA methyl esters were analyzed by gas chromatography (HP 6890series GC system; Agilent Technologies, Palo Alto, CA, USA). The gas chromatograph was equipped with an automatic on-column injector (HP 7673) (split ratio 4.325:1), a capillary column of 30 m × 320 μm inner diameter, 0.25 μm film thickness (Omegawax; Supelco 4-293-415) and a flame ionization detector. Helium was used as the carrier gas. The oven temperature was set at 86 °C, 4 min isothermal, 10 °C min−1 to 210 °C, 15 min isothermal. The detector was set at 250 °C. Chromatograms were recorded with a data system integrator (HP Chemstation; Hewlett-Packard, Palo Alto, CA, USA). Identifications of peaks were made by comparison of the retention times with those of a standard (mix of pure FAs from Sigma-Aldrich, Broendby, Denmark and Merck & Co Inc., NJ, USA) run under identical conditions. The quantity of FAs in each sample was calculated from the known quantity of internal standard by comparing the area under the curve of each FA methyl ester with that of 17:0 methyl ester.
Conjugated and free BAs in fecal samples were quantified by reversed-phase high-performance liquid chromatography with pulsed amperometric detection as outlined by Dekker et al.37 The freeze-dried samples were mixed using a Shaker VXR vibrax (IKA-Werke, Staufen, Germany) at 1500 r.p.m. for 30 s and subsequently centrifuged at 5000 g for 10 min. The supernatant was passed through a nylon syringe filter membrane (Cameo 17N-DDR02T17NB) before injection onto the high-performance liquid chromatography. The chromatographic conditions used are described by Knarreborg.38
Urine volume and density were measured and samples were stored at −20 °C until further analyses. Urinary calcium concentration was measured using atomic absorption on a Spectra AA-200 (Varian, Victoria, Australia). The CVintra% was 2.1 and the CVinter% was 2.9. Urinary nitrogen content was measured using the Dumas method with a nitrogen analyzer (NA1500; Carlo Erba Strumentazione, Milano, Italy). The CVintra% was 0.4 and the CVinter% was 1.9.
Samples of the two test diets, in which butter was not included, were homogenized and freeze-dried. The butter in the diets had to be omitted from the homogenization step, as it had previously been found impossible to distribute butter evenly in the homogenate using this method. Samples of the butter were analyzed separately for fat and FA content, whereas the calcium and energy contents in the butter were estimated using Dankost 3000.30 The fat, FA and energy content of the butter is incorporated in the reported values for the diets. Energy, calcium and fat content, and FA composition of the test diets were measured in the same way as the content and composition of the fecal samples.
It was estimated that a minimum of 10 subjects was required to attain a statistical power of 1-β=0.90, assuming a difference in fecal fat excretion of 5 g d−1 between diets and a within-subject standard deviation of 3 g d−1, based on data from the previous study from our group.19
Data were analyzed using Statistic Analysis Package, SAS version 8 (SAS Institute, Cary, NC, USA). Data are reported as mean±s.e.m. unless otherwise indicated. Statistical significance was set at P<0.05.
The difference in fecal fat, FA and energy content between the two diets was analyzed with and without adjustment for intake as the two diets, contrary to intentions, turned out not to have equal fat, FA and energy content. Without adjustment, Student's t-tests for paired observations were performed. Analyses with adjustments were performed by analysis of covariance, where ‘subject’ was included as a random variable. As no statistically significant effects of ‘sex’ and of ‘sequence’ were found, data from all subjects and from the two sequences were combined. Least square means were used to estimate the adjusted means.
Differences between means for the urinary parameters, transit time, fecal calcium excretion, fecal bile salt excretion and fecal weight were analyzed by the Student's t-test for paired observations.
Differences between the two diets in body weight and blood pressure measurements were analyzed by analysis of covariance, with the values from after the diet periods as dependent variables and the values from before the diet periods as covariates. For blood pressure, weight change was also included as a covariate. ‘Subject’ was included as a random variable. Differences between the values measured before and after the diet periods were analyzed by the Student's t-test for paired observations.
All 11 subjects (five men and six women) completed the two diet periods. Baseline characteristics of the subjects were as follows: age: 33 years (range 25–47); weight: 85.3±11.3 kg (mean±sd); height: 175±12 cm and BMI: 27.6±1.6 kg/m2. The subjects' estimated energy requirement was 11.9±2.5 MJ d−1 and their habitual calcium intake was 744±442 mg d−1.
Urine collections from one subject were missing. A few of the subjects reported having omitted certain food items from the diet plan, and a single subject reported having eaten additional food. The individual nutrient intake was adjusted for self-reported deviations from the diet plan using the Dankost 3000 dietary assessment software (Danish Catering Center).30
Fecal and urinary calcium excretion was significantly higher on the high-Ca compared with the low-Ca diet (Table 2). The combined fecal and urinary calcium excretion corresponded to approximately 96% of intake on the high-Ca and 105% on the low-Ca diet.
Fecal excretion of fat and energy
The fecal excretion of fat and energy was significantly higher on the high-Ca than on the low-Ca diet. On the high-Ca diet, the fecal fat excretion increased more than twofold compared with the low-Ca diet (11.5±1.4 vs 5.4±0.5 g d−1; P<0.0001). The difference in total fecal fat excretion between the diets could on average explain 91% of the difference in energy excretion.
As the dietary energy and fat intake, contrary to our intentions, was higher on the high-Ca diet than on the low-Ca diet (as presented in Table 3), this was adjusted for in analyses of covariance. The differences in fecal excretion of fat and energy between the diets were still highly significant when adjusted for intake. The means and the means adjusted for intake for the fecal excretions of fat and energy are listed together with the dietary intake in Table 3, where excretion as percentage of intake is also presented.
Fecal FA composition
For total FA, SFA, MUFA and PUFA and the main FAs (palmitic, stearic, oleic and linoleic acid), fecal excretions were significantly higher on the high-Ca than on the low-Ca diet. Adjusting for intake did not affect the results (Table 4). Fecal output of FAs other than palmitic, stearic, oleic and linoleic acid also increased during the high-Ca diet period, but they represented quantitatively only a small fraction of the total FA excretion (data not shown).
A comparison of the excretions expressed as percentage of intake gives an estimate of which FA group was affected most by the diets. SFA was significantly less affected by dietary calcium than MUFA (95% CI for the difference between diets in fecal excretions as % of intake: (3.2; 5.7) for SFA (6.6; 12.5) for MUFA and (3.9; 8.5) for PUFA).
Fecal bile acids
Total fecal excretion of BA did not differ in the two diet periods (Table 5) and neither did the excretion of primary and secondary BA (data not shown). However, the degree of conjugation was significantly higher on the high-Ca than on the low-Ca diet, with the fecal excretion of both taurine- and glycine-conjugated BA being greater on the high-Ca diet.
Fecal dry weight, fecal wet weight and transit time
Transit time was not significantly affected by diet (40.4±3.7 vs 40.7±3.3 h on the high-Ca and the low-Ca diets, respectively). Although fecal wet weight only tended to differ significantly between diets (236±38 vs 197±23 on the high-Ca and the low-Ca diets, respectively; P=0.0503), fecal dry weight was significantly higher on the high-Ca than on the low-Ca diet (55±7 vs 44±4 g d−1; P=0.0045).
Body weight, blood pressure and physical activity
No significant difference in body weight change or blood pressure was found between the high-Ca and the low-Ca diets (Table 6). However, small but significant weight losses were observed during both diet periods, and there was a significant drop in systolic blood pressure on the high-Ca diet.
The subjects' physical activity patterns were similar in the two diet periods, as assessed by self-recorded exercise diaries.
Compliance with the study diets
To assess compliance with the diet plans, both calcium excretion and urinary nitrogen excretion were determined. Nitrogen in urine was not significantly different between the diets (11.7±2.5 g d−1 on the high-Ca diet vs 11.6±2.9 g d−1 on the low-Ca (n=10)). These amounts of nitrogen corresponded to approximately 73 g protein (N × 6.25), which corresponded to 82% of the intake for both diets.
This study evaluated the short-term effect of increasing the intake of calcium from low-fat dairy products on quantitative and qualitative fecal fat loss. Our major finding is that a diet high in dairy calcium (∼2300 mg d−1) increased fecal fat excretion substantially (from 5.4 to 11.5 g d−1), with a corresponding increase in fecal energy excretion, compared with a low-calcium diet (∼700 mg d−1). Analyses of the FA composition of the fecal fat revealed that all major FAs consumed were excreted in greater amounts on the high-Ca diet.
By showing that calcium from low-fat dairy products enhances the fecal excretion of fat and energy, we confirmed the previous findings from our group.19 We found a 6.1 g d−1 difference in fecal fat between the two diet periods, which is comparable with the 8.2 g d−1 difference found in the study by Jacobsen et al.19 In both studies, the findings were validated by showing corresponding changes in the fecal energy excretion, which was measured by an independent technique. Adjusting for fat and energy intake did not affect the results notably.
To our knowledge, only two other randomized controlled studies in humans have examined the effect of calcium from dairy products on fecal fat excretion.23, 24 Govers et al.23 observed a 2.6 g d−1 increase in fecal fat excretion when the calcium intake from milk products in a double-blinded fashion was increased from ∼700 to 1800 mg d−1. Boon et al.24 found fecal fat excretions of 4.8, 7.2 and 7.5 g d−1 on high-protein diets with 400, 1200 and 2500 mg d−1 calcium from dairy products, respectively, but differences between diets did not reach statistical significance (n=10; P=0.159).
In this and the earlier study conducted by our group, the effect of increasing the intake of low-fat dairy products on fecal fat excretion was examined and not the effect of calcium per se. Therefore, we cannot rule out that calcium from other sources could have been equally effective in increasing the excretion of fecal fat. Boon et al.24 compared the effect of calcium from calcium carbonate with the effect of calcium from dairy products, but the study was underpowered to detect significant differences. Some investigators have examined the effect of calcium in a supplemental form on fecal excretion of fat and have found slightly smaller effects than we found,20, 21, 22, 25 whereas two studies have found no significant effect.39, 40
Calcium from dairy sources might be more effective than supplemental calcium in augmenting the fecal excretion of fat. In a recent meal study by our group, we found a negative impact on postprandial blood lipid response, indicative of decreased lipid absorption, in subjects consuming dairy products and not supplemental calcium.41 Other components of dairy products might be responsible for the possible differential effects between calcium and dairy products on fat absorption. Bioactive components in milk products may act either independently or synergistically with calcium. However, components responsible for increasing fecal fat loss have not yet been identified.
In this study, the fecal fat excretion was increased by 6.1 g d−1 on the high-Ca diet compared with the low-Ca diet, corresponding to a daily energy loss of ∼230 kJ. This could potentially induce a weight loss of 2.5 kg per year as described by Jacobsen et al.,19 although this estimate does not take into account that regulatory adjustments in energy expenditure may counterbalance some of the effect. In comparison, pharmacological treatment with orlistat has been shown to increase fecal fat excretion from baseline by 16 g d−1,42 with 66% of subjects reporting gastrointestinal adverse events. In our study, no adverse events were reported, probably due to the fact that fat bound by dairy calcium is in the form of semisolid soap, not oil.
We analyzed the FA composition of the total fecal fat to see whether the absorption of some FAs was more affected than the absorption of others. We found that the excretion of SFA, MUFA and PUFA was significantly higher on the high-Ca diet than on the low-Ca diet. When fecal excretions of SFA, MUFA and PUFA were expressed as percentages of intake, the difference between diets was greater for MUFA than for SFA. Contrary to our findings, others have speculated that the excretion of SFA is especially affected by increases in calcium intake due to a lower solubility of SFA calcium soaps.20, 25
The degree to which different FAs are excreted in the feces will potentially depend on the triacylglycerol structure of the dietary fat, as the positional distribution of the FAs on the dietary triacylglycerol is a determinant of which FAs are made available for soap formation. FAs in the sn-1 and sn-3 position are more susceptible to binding by calcium as they are hydrolyzed from the triacylglycerol molecule by the action of lipase during digestion in the intestinal lumen. Contrastingly, 75% of the FAs in the sn-2 position are conserved as monoacylglycerols up until absorption and are thus not present as free FAs in the intestinal lumen during absorption.43
The high-Ca diet in this study was high in dairy products (yogurt, milk, cream and butter) and thereby had a higher content of butter fat than the low-Ca diet. Butter fat is rich in SFA (68%) and the sn-2 position is dominated by SFA (73%).43 The choice of dairy products as a major fat source in our study could thus explain why SFA was not the FA fraction primarily affected by dietary calcium. In the two studies, where an effect mainly on the SFA was observed, the dietary fat source was primarily beef tallow25 or cocoa butter.20 These dietary fats are high in SFAs, though not as high as butter fat. However, the SFAs occupy the sn-2 positions only to a minor extent, which are primarily occupied by MUFA.43 The different sn-2 FA compositions of the dietary fat sources in the studies could thus be an explanation for the observed differences with respect to which FA fraction was affected the most.
It has been suggested that fat digestion decreases on high-calcium diets because calcium, together with phosphate, binds and precipitates BAs in the upper small intestine, thereby rendering less BAs available for micelle formation. If BAs are precipitated in the small intestine, an increase in fecal BA is to be expected. In this study, we did not observe an effect of calcium on total fecal BA excretion, but only an increase in the degree of conjugation. Our results thus do not support the findings of others who observed increases in fecal BA excretion of between 11 and 49% with high-calcium diets.18, 23, 39 All of these studies observed a concomitant increase in fecal phosphate, supportive of a role of phosphate in the precipitation of BAs. We did not measure fecal phosphate excretion. In accordance with our findings, Denke et al.25 observed no effect of supplementing with 2200 mg d−1 calcium (as calcium citrate malate) on fecal BA. However, in contrast to the methods used above, the high-performance liquid chromatography method used in the present experiment allows direct determination of conjugated and unconjugated, as well as primary and secondary, BA. Thus, the increased proportion of conjugated BA in the high-Ca diet has not been described before.
We employed two indirect measures of compliance: the calcium balance and the recovery of nitrogen in urine. We found that fecal and urinary calcium excretion combined corresponded to approximately 96% of intake on the high-Ca and 105% on the low-Ca diet. The fact that the total excretion of calcium in urine and feces hereby approximately balanced out the calcium intake was indicative of good compliance with the diet plans with respect to calcium.34 In addition, we found that the urinary nitrogen was indicative of a very good compliance with the diet plan with respect to protein intake, as nitrogen corresponding to 82% of the protein intake was found in both diet periods. It has previously been found that under normal circumstances, ∼81% of ingested protein is excreted in urine as nitrogen.35
We assessed the subjects' transit time by the use of transit markers. The mean transit time of ∼40 h in both diet periods indicated that the study design was appropriate, with collection of feces starting on the third day of each diet period.
In summary, we found a highly significant increase in the fecal excretion of fat and energy when the intake of calcium from dairy products was increased by 1600 g d−1. Increasing dietary calcium intake increased both SFA, MUFA and PUFA when dairy products were the main fat source. The fecal excretion of BAs was not affected by calcium content of the diet.
We are grateful to the laboratory and kitchen staff at the Department of Human Nutrition, especially I Timmermann, C Kostecki and Y Rasmussen, for their assistance. JK Lorenzen, NT Bendsen, A-L Hother and A Astrup designed the study. NT Bendsen and A-L Hother were responsible for collection of data and for analyzing the dietary and fecal samples. M Würtz is gratefully acknowledged for assistance with fat and FA analysis and so is L Hymøller for the HPLC part of the BA analysis. NT Bendsen and A-L Hother performed the data analysis. All authors participated in the discussion of the results and commented on the paper. The study was supported by the British Broadcasting Corporation (BBC).
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Conflicts of interest
A Astrup is a member of Arla Nutrition Advisory Board, The Global Dairy Platform, and receives an honorarium for each board meeting. NT Bendsen, A-L Hother, SK Jensen and JK Lorenzen have no conflicts of interest.