Reduced postprandial energy expenditure and increased exogenous fat oxidation in young woman after ingestion of test meals with a low protein content
© Petzke and Klaus; licensee BioMed Central Ltd. 2008
Received: 02 June 2008
Accepted: 17 October 2008
Published: 17 October 2008
Macronutrient composition of diets can influence energy balance in humans. We tested the hypothesis whether low protein content in single meals may induce lower values of energy expenditure (EE) and fat oxidation (FO) as compared to adequate protein content.
Indirect calorimetry was combined with a breath test using naturally 13C-enriched corn oil to differentiate between postprandial exogenous and endogenous FO. Young women ingested single meals containing either 3.9% (low protein, LP) or 11.4% (adequate protein, AP) of total energy (~3100 kJ) as protein.
Postprandial EE was 160 kJ/6 h lower (p < 0.01) after LP meals and diet induced thermogenesis (DIT) increased less (p < 0.001) as compared to AP meals. Total postprandial FO was not significantly different between meals (~17 g/6 h). However, exogenous postprandial FO was significantly (p < 0.01) higher (4.28 ± 1.57 g/6 h) after exposure to LP meals as compared to AP meals (1.87 ± 1.00 g/6 h). Less than 10% of ingested fat (50 g) was oxidized in the postprandial phase. The overall postprandial fat balance was approximately + 33 g.
Breath tests using naturally 13C-labeled corn oil mirror exogenous FO. Low protein meals resulted in reduced postprandial EE and increased exogenous FO as compared to adequate protein meals without differences in total FO.
Obesity is an increasing health problem in both developed and developing countries . Genetic predisposition, physical inactivity, and the consumption of high-energy dense foods are risk factors for obesity . It was shown that diet composition influences energy storage in the body which supported the view that macronutrients may play a role in the development of obesity. However, the results are contradictory and it remains unclear what the relative importance and metabolic advantages of protein, carbohydrate and fat are [3, 4]. Therefore, studies of in vivo effects of macronutrients on body fat storage and metabolism could enhance our understanding of the aetiology of obesity and improve methods for body-weight management.
In order to achieve a reduction in body fat, weight reducing diets must attain a negative energy or fat balance. An increase of the protein content of diets at the expense of carbohydrates has brought such an advantage [4–6]. Fat oxidation (FO) was shown to depend on dietary composition, being higher after a relatively high protein meal as compared to a balanced diet . Furthermore, there is increasing evidence of higher energy expenditure (EE) following protein ingestion as compared to carbohydrates or fat [8–12]. However, the mechanisms for the influence of dietary protein on energy expenditure are not clear and controversially discussed [6, 13, 14].
The combination of indirect calorimetry with isotope techniques is an effective tool for the examination of the oxidation of endogenous and ingested nutrients in vivo [15–19]. As demonstrated in recent reports 13CO2 breath tests are now widely used in metabolic studies of nutrients using stable isotopic labeled precursors [20, 21]. 10 to 30% of ingested fat were reported to be oxidized postprandially in subjects exposed to different experimental conditions usually using fat supplemented with 13C-labeled fatty acids [15–17]. Only few studies were published using 13C-labeled triglycerides as tracers . However, there are no reports in the recent literature available using non supplemented naturally enriched fat such as corn oil to study FO. The use of uniformly labeled naturally enriched fat in metabolic studies may have the advantage of avoiding artefacts due to possible differences in metabolism of artificially with stable isotopes labeled precursors as it was shown e. g. for crystalline amino acids compared to protein-bound amino acids . Therefore, we examined in a pilot study the effects of different test meals with low or adequate protein content and an equal fat content on postprandial EE and substrate oxidation. We hypothesized that an exposure of subjects to test meals with lower protein content may cause reduced rates of EE and FO with possible consequences on energy balance. Exogenous FO was measured using corn oil naturally enriched in 13C-content as the only fat component.
General characteristics of the study participants
Mean (n = 6) ± SD
25.5 ± 2.6
59.8 ± 8.3
1.7 ± 0.1
20.6 ± 2.5
Baseline δ13C in breath CO2 [‰ vs. PDB]
-25.8 ± 0.7
Composition and sources of test meals
δ13C [‰ vs. PDB]5
Skimmed milk powder1
-28.2 ± 0.7
-16.1 ± 0.2
-26.9 ± 0.2
-27.8 ± 0.2
Macronutrient composition of test meals1
Energy content (kJ)
Protein (% total energy)
Total fat (g)
Total fat (kJ)
Total fat (% total energy)
Carbohydrate (% total energy)
Study protocol and calorimetry
Studies were performed after an overnight (12 h) fast. All participants took part twice and consumed both test meals randomly in a distance of 2 days in between. All subjects underwent measurements of EE using an open-circuit indirect calorimeter with a ventilated-hood system (Deltatrac™ II, Datex Instrumentarium Corp., Helsinki, Finland). The system was calibrated daily according to the instructions using 5% CO2 in O2 calibration gas (QUICK CAL™, GE Healthcare Finland Oy, Helsinki). After a run-in period of 30 min, energy expenditure (EE) was determined between 08:00 and 09:00 within 30 min. Then, subjects ingested the test meals over a period of 10 min and postprandial EE was measured three times after 30, 150, and 270 min of the study protocol. Breath samples were collected 5-fold at baseline (-10 min) and twice every 30 min for a period of 6 h after the meal into tubes (10 mL Exetainer®, Labco Ltd., Buckinghamshire, U.K.) for measurement of breath13CO2 enrichments. The bladder was emptied before the study. During the experimental period within a total time interval of 360 min, complete urine samples were collected for the assessment of nitrogen excretion. Nitrogen content in urine was determined by Kjeldahl method (Kjeldatherm-Turbosog-Vapodest 45, C. Gerhardt GmbH & Co. KG, Bonn, Germany). For each measurement of EE the first 5 to 10 min were discarded to allow subjects to adapt to the measurement procedure, and data from the remaining 20 min were averaged and used to calculate EE and substrate oxidation based on oxygen consumption, carbon dioxide production, and urinary nitrogen excretion.
Stable isotopic analysis
where Rstandard = [13C]/[12C] = 0.0112372 for PDB that has been assigned a δ13C value of 0.0‰. Rsample is derived from the ratios of the mass spectrometer ion currents ranging from m/z 44 to m/z 46.
Calculations and statistics
Baseline measurements before ingestion of test meals (n = 6)
218 ± 12
230 ± 13
0.87 ± 0.04
0.86 ± 0.08
2.73 ± 0.66
2.73 ± 0.66
2.72 ± 1.53
3.05 ± 2.53
10.7 ± 2.9
8.65 ± 4.41
where QexFO is the quantity of ingested fat oxidized, δb is the baseline breath 13CO2 abundance (‰ δ13C vs. PDB) δt is the breath 13CO2 abundance at time t post meal ingestion, δt-1 is breath 13CO2 abundance at any other time after t, δs is the 13C abundance of labeled fat ingested, QCO2 is the quantity of CO2 expired in breath (moles) for the time interval in question, MW is the average molecular weight (866 g/mol) of ingested corn oil triglycerides, and n (= 55) is the number of carbon atoms in corn oil triglyceride molecule. 0.56 is the acetate correction factor to account for label fixation that might occur at any step between the entrance of labeled acetyl-CoA into the tricarboxylic acid cycle until the recovery of label in breath CO2 .
Data are reported as means ± SD. Comparison of means was performed with two-sided paired t-test. Pearson correlation coefficients were calculated to determine the relationship between selected parameters. Differences with p < 0.05 were considered statistically significant. WinSTAT® (vers. 1999.2, R. Fitch software, Staufen, Germany) was used to compute statistical analysis.
The influence of the test meals on postprandial energy expenditure and substrate oxidation (n = 6)
Postprandial EE (kJ/6 h)
1356 ± 69
1516 ± 66
DIT (kJ/6 h)1
46.5 ± 5.7
134.6 ± 34.3
9.0 ± 1.1
25.9 ± 6.6
0.87 ± 0.04
0.87 ± 0.03
Change in RQ
-0.01 ± 0.06
0.01 ± 0.05
Postprandial PO (g/6 h)
14.9 ± 4.20
18.0 ± 4.2
Change in PO (g/6 h)1
-1.50 ± 1.23
1.55 ± 1.29
Postprandial CO (g/6 h)
55.6 ± 17.8
58.7 ± 14.1
Change in CO (g/6 h)1
-8.70 ± 27.65
6.77 ± 25.34
Postprandial FO (g/6 h)
16.9 ± 7.5
16.8 ± 8.8
Change in FO (g/6 h)1
0.55 ± 7.91
-1.50 ± 7.00
Endogenous postprandial FO (g/6 h)2
12.6 ± 7.8
14.9 ± 9.2
Exogenous postprandial FO (g/6 h)3
4.28 ± 1.57
1.87 ± 1.00
Contribution of exogenous postprandial FO of energy expenditure (kJ/6 h; % of postprandial EE)
In the present pilot study we examined the effect of single ingestions of different test meals with either low or higher but adequate protein content on postprandial energy metabolism and FO in young woman. This was performed to test our hypothesis that an exposure to low protein containing meals may lead to reduced rates of EE and FO as compared to adequate protein meals which may have consequences on energy balance. Our results confirmed that dietary protein may influence postprandial EE and DIT. The postprandial EE was significantly higher in woman after exposure to the AP meal (protein content of 11.4% of energy) as compared to the LP meal (protein content of 3.9% of energy). The value of postprandial EE of young woman was approximately by 160 kJ/6 h higher after exposure to the AP meal as compared to the LP meal. This accounts to approximately 5% of the energy content of test meals. Therefore, the present results confirmed former studies which have shown that protein can induce a higher thermic effect compared to other macronutrients and that high protein diets have a higher potential to increase EE than low protein diets [10, 32, 33]. However, data in literature are not uniform. It was shown under conditions of energy-restriction that a short-term replacement of dietary carbohydrate with protein did not increase the thermic effect of food to facilitate weight loss in type 2 diabetes patients . Moreover, overfeeding of either low- or high-protein diets were found to increase DIT as compared to AP diets . Although, habitual energy intake of young woman was not evaluated in our present pilot study, we assumed no restriction or excess in food energy consumption before test meal consumption. In future studies of postprandial effects of protein consumption an evaluation of the role of previous energy intake will be necessary because food energy deficiency was supposed to prevent thermic effects of dietary proteins . The mechanisms regulating thermic effects after consumption of high protein diets remain to be elucidated. However, based on the prevention of thermic effects in energy deficient states a role of mitochondrial metabolism in modifying energy efficiency may be supposed .
In addition, it has been shown that subjects consuming high protein diets (30% of energy) for 4 d exhibited an increased EE and higher rates of FO resulting in lower body fat mass when fed at energy balance and compared to diets containing an adequate protein content (10% of energy) [33, 36]. In the present study we did not find differences in total FO in subjects exposed to LP or AP test meals. Interestingly, exogenous postprandial FO was significantly lower in subjects exposed to AP meals compared to LP meals. It was shown in former studies that net lipid oxidation is dependent on short-term changes in dietary intake, being significantly higher after a high protein meal compared to a meal relatively balanced in macronutrient composition . The reason for lower rates of exogenous FO after exposure to AP meals compared to LP meals is not clear. However, lower rates of gastric emptying after administration of diets containing higher proportions of milk proteins can not be excluded and have to be evaluated in future studies . Taken together, it seems that although able to exhibit higher rates of exogenous FO, meals with a relatively low protein content are not advantageous compared to adequate protein meals with respect of lowering total energy or fat balance.
The measurement of enrichment in expired 13CO2 is widely used to determine nutrient oxidation rates using different infusion techniques or oral consumption of 13C-labeled substrates. Mostly substrates are applied which were exogenously labeled with 13C as fatty acids to enrich the precursor pools by supplementation to fat of test meals to increase the outcome of 13CO2 enrichment in breath samples. However, fatty acids may be metabolized differently than nutrients like fat usually consumed with complex diets. Therefore, intrinsically or naturally enriched tracers should be preferred. Analytical techniques are now sensitive enough to detect relatively small changes of enrichments in the range of natural abundances. Corn oil is naturally enriched (about -16 ‰ δ13C) due to discriminatory accumulation of 13C during photosynthesis. Naturally 13C-labeled triglycerides from corn oil are absorbed and cleaved by lipolysis to 13C-labeled fatty acids and glycerol before incorporation into endogenous fat deposits or subsequent mitochondrial β-oxidation, which results in the production of 13CO2 in breath. Therefore, naturally 13C-enriched corn oil can be used to examine exogenous FO when the subject's habitual diet and test meal components do not contain other corn based products. In the present experiment we have shown that a supplementation of corn oil with 13C-fatty acids was not required. This avoids possible artefacts due to different absorption and metabolism of fatty acids as compared to triglycerides normally available from dietary fat. In addition, no additional assumptions are necessary with respect to not uniformly labeled fatty acids usually supplemented as a tracer. The calculation of exogenous postprandial FO was possible although the mean increase of δ13C values over baseline in breath CO2 amounted to only 1 to 2 ‰ vs. PDB. The method permitted a significant differentiation of effects due to differences in test meal composition on FO. However, it was an essential preposition that baseline values of 13CO2 enrichment did not change during experimental period which we have tested using sunflower seed oil instead of corn oil [Unpublished observations by Petzke].
We have shown that corn oil naturally enriched with 13C can be used to evaluate postprandial FO in vivo. Furthermore, we provide evidence that the amount of dietary protein in relation to other macronutrients may influence postprandial exogenous and endogenous FO the consequences and mechanisms of action of which has to be examined in future studies.
Body mass index
Diet induced thermogenesis
Delta value over baseline
Elemental analyser/isotope ratio mass spectrometer
PeeDee Belemnite Limestone carbonate
Petra Albrecht is gratefully acknowledged for excellent technical assistance.
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