Obesity is an increasing health problem in both developed and developing countries 1. Genetic predisposition, physical inactivity, and the consumption of high-energy dense foods are risk factors for obesity 2. 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 34. 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 456. Fat oxidation (FO) was shown to depend on dietary composition, being higher after a relatively high protein meal as compared to a balanced diet 7. Furthermore, there is increasing evidence of higher energy expenditure (EE) following protein ingestion as compared to carbohydrates or fat 89101112. However, the mechanisms for the influence of dietary protein on energy expenditure are not clear and controversially discussed 61314.

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 1516171819. As demonstrated in recent reports ¹³CO₂ breath tests are now widely used in metabolic studies of nutrients using stable isotopic labeled precursors 2021. 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 151617. Only few studies were published using ¹³C-labeled triglycerides as tracers 22. 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 23. 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.

EE and DIT expressed in absolute terms (kJ/6 h) or as percentage of energy in test meals were significantly lower after ingestion of LP meals compared to AP meals (Table 5). There was no change in RQ following both test meals and no significant difference in RQ between different test meals. The relatively high baseline RQ (Table 4) can not be satisfactorily explained. It could be that the overnight fasting period was not sufficient to deplete glycogen stores. Alternatively, the resting period of 30 min before measurement of the resting EE might not have been sufficient for young, healthy normal weight female subjects. As expected, a lower protein intake with the LP meal reduced postprandial PO in relation to fasting levels. On the other hand postprandial PO increased relative to fasting levels following AP meals. The change in PO between test meals was significantly different. Total postprandial CO was not significantly different (p > 0.05) after exposure to different test meals. Total postprandial FO was not significantly different between both test meals. However, postprandial FO slightly increased or decreased as compared to measurements during fasting after exposure to LP or AP meals, respectively. Interestingly, exogenous postprandial FO was significantly higher (p = 0.005) by about 2.4 g/6 h after ingestion of LP meals compared to AP meals. However, the total amount of oxidized exogenous fat during 6 h was relatively low (less than 10% compared to ingested fat mass). On the other hand, endogenous postprandial FO was not significantly different between young woman ingesting LP or AP test meals due to high individual variations. The overall postprandial fat balance was approximately + 33 g. Interestingly, DIT positively correlated with PO (R² = 0.739, p = 0.011), and exogenous FO negatively correlated with change in PO (R² = 0.532, p = 0.037).

Baseline breath δ¹³CO₂ values (Table 1) were similar to those of C₃ plant based nutrients (Table 2, 22). In response to test meal ingestion it was evident that DOB values of breath ¹³CO₂ progressively increased in all subjects (Figure 1). A peak value of ¹³C enrichment in breath ¹³CO₂ was not reached during 6 h of the study protocol. DOB values increased within 6 h up to 1 or 2 ‰ δ¹³C after ingestion of AP- or LP-meals, respectively. The δ¹³C values in breath CO₂ of young woman were significantly different (p < 0.05) from 180 min and thereafter comparing between the exposure to LP and AP meals.

The time course of cumulative exogenous postprandial FO calculated from δ¹³CO₂ breath values is shown in Figure 2. Cumulative exogenous postprandial FO based on ¹³CO₂ production from corn oil was significantly higher (p < 0.05) as of 270 min and thereafter of young woman exposed to the LP meal as compared to AP meal.

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 103233. 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 34. Moreover, overfeeding of either low- or high-protein diets were found to increase DIT as compared to AP diets 12. 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 35. 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 35.

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) 3336. 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 7. 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 37. 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.

AP: Adequate protein; BMI: Body mass index; CHO: Carbohydrate oxidation; DIT: Diet induced thermogenesis; DOB: Delta value over baseline; EE: Energy expenditure; EA/IRMS: Elemental analyser/isotope ratio mass spectrometer; FO: Fat oxidation; LP: Low protein; PDB: PeeDee Belemnite Limestone carbonate; PO: Protein oxidation; RQ: Respiratory quotient.

The authors declare that they have no competing interests.

KJP has contributed to the design of the experiment and conducted and directed the sample and data analysis. Furthermore, he was involved in the writing and editing of this manuscript. SK directed the research and contributed to the preparation of the manuscript.