The thermic effect of feeding (TEF: increase in energy expenditure following acute energy intake) is an important physiological determinant of total daily energy expenditure and thus energy balance. Approximately 40% of TEF is believed to be mediated by sympathoadrenal activation and consequent β-adrenergic receptor stimulation of metabolism. In sedentary adults, acute administration of ascorbic acid, a potent antioxidant, augments the thermogenic response to β-adrenergic stimulation. We hypothesized that acute ascorbic acid administration augments TEF in sedentary overweight and obese adults. Energy expenditure was determined (ventilated hood technique) before and 4 h after consumption of a liquid-mixed meal (caloric equivalent 40% of resting energy expenditure (REE)) in 11 sedentary, overweight/obese adults (5 men, 6 women; age: 24 ± 2 years; BMI: 28.5 ± 1.0 kg/m2 (mean ± s.e.)) on two separate, randomly ordered occasions: during continuous intravenous administration of saline (placebo control) and/or ascorbic acid (0.05 g/kg fat-free mass). Acute ascorbic acid administration prevented the increase in plasma concentration of oxidized low-density lipoprotein in the postprandial state (P = 0.04), but did not influence REE (1,668 ± 107 kcal/day vs.1,684 ± 84 kcal/day; P = 0.91) or the area under the TEF response curve (33.4 ± 2.4 kcal vs. 30.5 ± 3.6 kcal; P = 0.52) (control vs. ascorbic acid, respectively). Furthermore, acute ascorbic acid administration had no effect on respiratory exchange ratio, heart rate, or arterial blood pressure in the pre- and postabsorptive states (all P > 0.64). These data imply that the attenuated TEF commonly observed with sedentary lifestyle and obesity is not modulated by ascorbic acid-sensitive oxidative stress.
The thermic effect of feeding (TEF: the increase in energy expenditure following acute dietary intake) accounts for ∼10% of total daily energy expenditure (1). Thus, it is a significant component of energy balance and therefore, may have the potential to contribute to changes in body weight/composition over time.
It has been established that TEF is lower in sedentary adults compared with their habitually exercising counterparts (2,3,4). This may be due in part to greater thermogenic β-adrenergic receptor (β-AR) responsiveness in habitual exercisers (5). Approximately 40% of TEF is thought to be mediated by activation of the sympathoadrenal system and subsequent β-AR stimulation of metabolism (6). A recent study demonstrated that both TEF and the thermogenic response to intravenous β-AR stimulation were greater in habitual exercisers compared with sedentary adults (3). Furthermore, TEF was positively associated with β-AR-mediated thermogenesis.
The lower thermogenic β-AR responsiveness of sedentary adults may be partially attributed to oxidative stress (5). Co-infusion of the powerful antioxidant ascorbic acid increased the thermogenic response to intravenous β-AR stimulation in sedentary adults such that it was no longer less than that of habitual exercisers. When the experiment was repeated in habitual exercisers, ascorbic acid had no effect on thermogenic β-AR responsiveness, presumably because habitual exercise is associated with lower tonic oxidative stress (7,8).
With this information as a background, we tested the hypothesis that acute administration of ascorbic acid augments TEF in sedentary adults who are overweight or obese, a population previously shown to have appreciably high levels of oxidative stress (9,10).
Methods and Procedures
We studied 11 adults (5 men and 6 women) (between 18 and 36 years of age). All were overweight (25.0 ≤ BMI ≤ 29.9 kg/m2) or obese (BMI ≥ 30.0 kg/m2). None of the subjects had performed any type of regular exercise during the previous 2 years and, compared with US population norms, all were at or below the 50th percentile for maximal oxygen uptake (VO2max), a measure of maximal aerobic exercise capacity, based on their age (11). Subjects were nonsmokers and were not regularly taking any medications or vitamin/antioxidant supplements. The nature, purpose, and risks of the study were explained to each subject before written informed consent was obtained. The experimental protocol conformed to the standards set by the Declaration of Helsinki and was approved by the Human Research Committee at Colorado State University.
Following screening procedures (body composition and aerobic fitness), subjects were studied during two mornings separated by an average of 10 days. TEF was determined on both days, once during intravenous administration of saline (100 ml delivered over 4.5 h) and once during intravenous administration of ascorbic acid dissolved in 100 ml of saline (0.05 g per kg fat-free mass; delivered over 4.5 h) in a single blind, randomized fashion. This dosing regimen has been previously used to increase plasma ascorbic acid concentration ∼15-fold above baseline (12) and increase thermogenic response to β-AR stimulation (5). Each session was performed under standardized conditions after a 12-h fast, 2-h abstention from water, and 24-h abstention from exercise. Subjects were studied under quiet resting conditions in the semi-recumbent position. Measurements were initiated between 0700 and 0900 hours in a dimly lit room at a comfortable temperature (∼23 °C). Females were studied during the early-follicular phase of the menstrual cycle.
TEF was determined using a modification of previously described procedures (2,3). Briefly, subjects were instrumented for measurement of heart rate (electrocardiogram) and blood pressure (automated device, Cardiocap 5; GE Datex-Ohmeda, Madison, WI), and a catheter was placed in an antecubital vein and kept patent with heparin. Following instrumentation, resting energy expenditure (REE) was measured over 45 min. The first 15 min were considered a habituation period after which intravenous administration of saline and/or ascorbic acid began. VO2 and carbon dioxide production (VCO2) were averaged each minute for 30 min using a custom built ventilated hood indirect calorimetry system (Nighthawk Design, Boulder, CO) that used a respiratory mass spectrometer (Perkin Elmer MGA 1100; MA Tech Services, St. Louis, MO) and an ultrasonic flow sensor (ndd Medizintechnik AG, Zürich, Switzerland), and was calibrated daily with precision-mixed gases. In our laboratory, the measurement of REE has a coefficient of variation of 3.3% and a test-retest r2 of 0.93. Following measurement of REE, subjects consumed a commercially available liquid-mixed meal (Ross Laboratories, Abbott Park, IL; 57% carbohydrates, 28% fat, and 15% protein). To standardize the stimulus for each individual, the administered caloric load was equivalent to 40% of REE, resulting in meal sizes ranging between 520 and 972 kcal. The caloric load was chosen because it represents ∼30% of total daily caloric requirements (assuming REE accounts for ∼75% of total daily energy expenditure (13)), and is reflective of a normal meal. Subjects consumed the liquid meal within 15 min. The TEF (i.e., the increase in energy expenditure above preprandial baseline levels during the postprandial period) was then measured for 4 h. Indirect calorimetric measurements were made for 20 min, each for a 30-min period, allowing the subject relief from the ventilated hood for 10 min of each half an hour, at which time postprandial blood samples (20 ml preserved with K3 EDTA + 5 ml preserved with EGTA/glutathione) were collected in chilled tubes for measurement of concentrations of plasma glucose, insulin, catecholamines, and oxidized low-density lipoprotein (LDL), a systemic marker of oxidative stress. TEF was calculated for each individual as the change (increase) in energy expenditure from baseline across each time point and as the area under the response curve (trapezoidal rule).
All blood samples were immediately placed on ice and centrifuged within 60 min of collection to isolate plasma. Plasma samples were stored at −80 °C until analysis. Plasma catecholamine concentrations were analyzed in duplicate using high-performance liquid chromatography. Enzyme-linked immunosorbent assays were used to measure, in duplicate, plasma concentrations of insulin (Millipore, Billerica, MA), glucose (Thermo Electron, Pittsburgh, PA) and oxidized LDL.
Fat mass and fat-free mass were measured using dual-energy X-ray absorptiometry (DXA-IQ, software version 4.1; Lunar Radiation, Madison, WI). VO2max was determined with a metabolic cart (Parvo Medics, Sandy, UT) during incremental treadmill exercise as previously described (14). Briefly, subjects walked/ran on a treadmill at an increasing grade until three of the following criteria were satisfied: volitional exhaustion (defined as an inability to continue), a heart rate within 10 beats/min of their age-related maximum (15), a plateau in the VO2-work rate relation, and a rating of perceived exertion >19 (ref. 16).
Because this was a within-subject, repeated-measures design energy expenditure was not adjusted for fat-free mass. Based on measures of skewness and kurtosis the data were normally distributed, thus differences in TEF during administration of saline and/or ascorbic acid were analyzed using two-way ANOVA (condition × time) with repeated measures (energy expenditure) and also using one-way ANOVA (area under the response curve). Similarly, differences in plasma markers were analyzed using two-way ANOVA with repeated measures. Multiple comparisons of factor means were performed using the Neuman-Keuls test.
The level of statistical significance was set at P < 0.05. Data are expressed as mean ± s.e.
Selected subject characteristics are displayed in Table 1. Subjects were overweight or obese (25.7 < BMI < 35.4 kg/m2), normotensive, and had normal fasting blood glucose and insulin concentrations.
Table 1. Selected subject characteristics
Ascorbic acid and oxidative stress
Basal oxidative stress, as reflected by baseline plasma concentration of oxidized LDL, was not different between trials (37.1 ± 1.4 U/l vs. 38.3 ± 4.4 U/l; P = 0.99; control vs. ascorbic acid, respectively). Following consumption of the liquid meal, oxidized LDL concentration increased in the control condition (Figure 1) but not in the ascorbic acid condition (condition × time interaction: P = 0.042).
REE was unaffected by ascorbic acid (1,668 ± 107 kcal/day vs. 1,684 ± 84 kcal/day; P = 0.91; control vs. ascorbic acid, respectively). Following feeding, energy expenditure was increased above baseline (P < 0.001; Figure 2) but this TEF response was unaffected by ascorbic acid, regardless of expression: absolute energy expenditure (P = 0.71) and area under the TEF response curve (P = 0.52). Respiratory exchange ratio (VCO2/VO2; Table 2), an indicator of substrate utilization, was not different between trials at baseline (0.81 ± 0.01 vs. 0.81 ± 0.02), increased in the postprandial state (P < 0.0001) but was not influenced by ascorbic acid (P = 0.55). β-ARs play an important role in cardiovascular regulation. To determine the influence of acute ascorbic acid administration on cardiovascular responses to feeding, we measured heart rate and arterial blood pressure (Table 2). Heart rate was increased above baseline (∼15 beats/min) in the postprandial state (P < 0.0001) but was unaffected by acute ascorbic acid administration (P = 0.81). Neither systolic nor diastolic blood pressure changed appreciably from baseline (P > 0.15), nor were they affected by ascorbic acid (P > 0.64).
Table 2. Cardiovascular and selected blood parameters in overweight and obese adults before and after consumption of a liquid-mixed meal (equivalent to 40% of resting energy expenditure) during acute administration of either saline (control) or ascorbic acid (0.05 g/kg fat-free mass)
Plasma concentrations of epinephrine and norepinephrine revealed that feeding increased the activity of the sympathoadrenal system (P < 0.001), but this activation was not influenced by acute ascorbic acid administration (P > 0.14; Table 2). Similarly, plasma concentrations of glucose and insulin were increased following feeding (P < 0.001), but neither was influenced by ascorbic acid (P > 0.90; Table 2).
Based on previous observations in sedentary adults of (i) decreased TEF (2,3,4) and decreased thermogenic responsiveness to β-AR stimulation (5); (ii) the attribution of 40% of the TEF response to β-AR stimulation (6); and (iii) augmented thermogenic response to β-AR stimulation with ascorbic acid (5), we have investigated the novel hypothesis that acute ascorbic acid administration augments TEF in sedentary overweight and obese adults. Contrary to our hypothesis, we found no beneficial effect of acute ascorbic acid administration on TEF.
Critical to our hypothesis is the rationale that acute ascorbic acid administration decreased oxidative stress and improved β-AR-mediated thermogenic responsiveness. With respect to this first point, acute administration of ascorbic acid prevented the increase in plasma-oxidized LDL concentration, a systemic marker of oxidative stress, during feeding (Figure 1). In further support of the effectiveness of our ascorbic acid-dosing regimen, several other studies using comparable acute dosing have also described decreased plasma markers of oxidative stress and/or augmented physiological function (12,14,17,18). Similarly, augmented β-AR function with comparable acute ascorbic acid administration has been reported in several separate studies (5,19,20). It is feasible that, in this study, despite decreased plasma-oxidized LDL concentration no corresponding decreases in oxidative stress occurred in tissues that contributed directly to the TEF response and β-AR function. Furthermore, it is also possible that TEF overall, and/or the β-AR component of TEF, is not modulated by ascorbic acid-sensitive oxidative stress.
Also critical to our hypothesis is the notion that β-ARs account for a significant proportion of the TEF response, and that by improving β-AR function TEF would be increased. Data from our current study might suggest that the β-AR contribution to TEF has been overestimated. This argument seems unlikely based on separate observations of appreciably attenuated TEF during β-AR blockade with propranolol (21) and during inhibition of sympathoadrenal stimulation of β-ARs with clonidine (22), in addition to positive statistical associations between TEF and β-AR-mediated thermogenesis (23). Conversely, one previous study reported no effect of propranolol on TEF in adult men (24), although in this study the small dose of propranolol was likely insufficient to elicit effective β-AR blockade (25), consequently the evidence for substantial β-AR contribution to TEF remains compelling.
An alternative explanation of our data is that β-AR function was improved with ascorbic acid; however, acute ascorbic acid administration may have decreased the feeding-induced activation of the sympthoadrenal system, resulting in decreased β-AR stimulation. This explanation is doubtful given the lack of effect of ascorbic acid on the catecholamine response to feeding (Table 2). Consistent with this observation, intravenous ascorbic acid administration does not influence skeletal muscle sympathetic nerve activity, as determined using microneurography, in young or older adults (12).
A second alternative explanation of our data relates to the thermogenic properties of insulin (26,27). The increase in circulating insulin following feeding is also thought to contribute to TEF (28,29). There is some evidence that insulin resistance is mediated in part by oxidative stress and that administration of antioxidants augment insulin sensitivity (30,31). Accordingly, it is possible that administration of ascorbic acid to our sedentary overweight and obese subjects improved insulin sensitivity and decreased circulating insulin concentration thereby influencing the overall TEF response. However, our data demonstrate that acute ascorbic acid administration had no effect on insulin (or glucose) before or after feeding.
During the control condition (saline administration), plasma concentration of oxidized LDL was increased following feeding (Figure 1). This observation is consistent with reports of endothelial dysfunction following feeding, particularly if the meal has a high fat content (32,33). This endothelial dysfunction is thought to be mediated through an oxidative stress mechanism, as endothelial function is preserved with prior ascorbic acid administration (34,35,36).
A potential limitation of this current study pertains to our 4-h measurement of TEF. Energy expenditure can remain elevated above baseline for >5 h after feeding (37,38,39,40). It is feasible that had we measured TEF over a longer duration we may have observed a significant effect of ascorbic acid administration, however given the barely detectable time-condition interaction over 4 h (P = 0.71) this seems unlikely. Moreover, it has been argued that measuring 70% of the TEF response is sufficient for detecting group or treatment differences (39,41). What is more, the initial 4 h following feeding is the period that appears to be most influenced by β-AR signaling (2). At the very least, our data suggest that over 4 h TEF is unaffected by acute ascorbic acid administration.
In this study, basal oxidative stress, as estimated by plasma-oxidized LDL concentration, was comparable to values reported for similar sedentary, young adults (14) but appeared lower relative to sedentary older adults (14) and adults with characteristics of the metabolic syndrome (42). Thus, it is plausible that acute ascorbic acid administration may favorably influence TEF in a population with greater basal oxidative stress. In a related point, we did not measure baseline plasma ascorbic acid concentration; the relatively low level of oxidative stress in our research participants may have been due in part to substantial daily intake of vitamin C from dietary sources.
Our acute administration of ascorbic acid did not influence TEF. The possibility that oxidative stress impairs TEF in a genomic-mediated manner (e.g., DNA damage) is worthy of consideration. This might imply that chronic rather than acute administration of ascorbic acid would augment TEF, however at this time we are unable to support or refute this idea confidently. Several previous studies have demonstrated that compared with acute intravenous administration, chronic oral administration of ascorbic acid leads to increases in plasma ascorbic acid concentration of a smaller magnitude (14,17), and fails to decrease oxidative stress (14) or modulate physiological (baroreflex) function (17). Alternatively, chronic administration of a cocktail of antioxidants has been shown to augment renal function, whereas acute administration had little effect (43). Clearly, a definitive answer will be obtained only through empirical investigation.
TEF accounts for a significant proportion of estimated total daily energy expenditure, thus the question of whether it could be augmented with acute ascorbic acid administration has obvious clinical importance. Although our data suggest that acute ascorbic acid administration may not be an effective mechanism to increase TEF, and thus favorably modify energy balance, this powerful antioxidant may still have a clinical application based on its ability to preserve postprandial endothelial function in at-risk populations (34,35,36).
In conclusion, based on sound rationale and previous independent scientific observations, we investigated the novel hypothesis that acute ascorbic acid administration would augment TEF in sedentary overweight and obese adults. Our data imply that the attenuated TEF commonly observed with sedentary lifestyle and the overweight state is not modulated by ascorbic acid-sensitive oxidative stress.
This research was supported by awards from the National Institutes of Health (NIA AG022053 and NIDDK 5 P30 DK057516-07) and the United States Department of Agriculture, Colorado Agricultural Experiment Station. We thank the General Clinical Research Center at Pennsylvania State University, Hershey, PA for assistance with plasma catecholamine analysis.