Research Design and Methods
We screened 47 individuals between 2008 and 2013 for this study; 15 healthy (based on physical examination and laboratory analysis, electrocardiography, and psychological assessment) but obese volunteers were admitted to our inpatient unit. Use of nicotine and illicit drugs was excluded by screening tests at admission, and none of the subjects were taking any medication. Three volunteers did not complete the study because of a family emergency, cellulitis of the right toe, and inability to continue CR; 12 volunteers were included in this analysis. General, anthropometric, and energy expenditure characteristics of the study population are provided in Table 1. Subjects were admitted for 77 ± 4 days to the inpatient unit of the Obesity and Diabetes Clinical Research Section of the National Institute of Diabetes and Digestive and Kidney Diseases in Phoenix, AZ (Fig. 2), where they were limited to primarily sedentary activity for the duration of the study. The research unit was kept at room temperature. Minor variability in days spent on the metabolic ward was the result of scheduling individual study procedures during baseline or after CR. Subjects were weighed in the same light clothes daily upon first awakening and were asked not to exercise for the duration of their stay. Volunteers were fully informed of the nature and purpose of the study, and written informed consent was obtained before admission. The experimental protocol was approved by the institutional review board of the National Institute of Diabetes and Digestive and Kidney Diseases.
Study design. Bomb refers to the bomb calorimetry of intake (food) and output (urine and feces). CR, 24h-EE assessment during caloric restriction; EB 1, 24h-EE assessment to determine weight-maintaining energy needs; EB 2, 24h-EE assessment in energy balance; EB 3, 24h-EE assessment in energy balance at new weight; FST, 24h-EE assessment during fasting; OF, 24h-EE assessment during overfeeding chamber; OGTT, oral glucose tolerance test; **At least 48 h at 100% WMD between the two 24h-EE assessments.
Study Overview. Baseline Metabolic Stabilization Period: Upon admission volunteers were provided a standard weight-maintaining diet (WMD) with 50%, 30%, 20% carbohydrate, fat, and protein content, respectively. Individual weight-maintaining energy needs were determined based on weight and sex. The WMD was provided throughout the baseline period (22 ± 4 days), except for four 24-h periods, which volunteers spent in the whole-room indirect calorimeter (details below). Bomb calorimetry (details below) of a duplicate WMD meal was performed 3 days/week to accurately determine energy intake. If needed, daily caloric intake was adjusted to maintain a stable body weight (±1%). Urine and stool were collected 3 days/week and the energy content lost through excretion was determined by combustion. Average daily urine and stool calories were extrapolated to the rest of the associated week. Diabetes was ruled out by a 2-h, 75-g oral glucose tolerance test (after a 12-h overnight fast) according to the American Diabetes Association diagnostic criteria. Body composition (fat mass [FM] and fat-free mass [FFM]) was estimated by total body dual-energy X-ray absorptiometry (DXA) (DPX-L; Lunar Radiation, Madison, WI). Percentage body fat, FM, and FFM were estimated as previously described.
CR (42 Days): Volunteers consumed a 50% calorie-reduced liquid diet (Ensure; Abbott Laboratories, Columbus, OH) based on their calculated weight-maintaining energy needs. Bomb calorimetry of duplicate meals was performed daily. Urine and stool were collected 3 days/week and the energy content was determined by combustion; 24h-EE was measured weekly, and spontaneous physical activity (details below) was assessed daily during the entire 6-week CR period. DXA scans were performed biweekly.
After CR: For 12 ± 2 days following CR, volunteers were fed a standard 100% WMD, based on their new weight. 24h-EE measurements, total-body DXA, and oral glucose tolerance tests were repeated. Duplicate bomb calorimetry was performed on WMD meals, urine, and stool 3 days/week.
Measurements. Energy Expenditure: 24h-EE was assessed in a large, open-circuit indirect calorimeter (respiratory chamber), as previously described. Sleeping energy expenditure was calculated from 24h-EE as an approximate surrogate for resting metabolic rate between 11:00 p.m. and 5:30 a.m.
To closely achieve energy balance during the 24h-EE assessment and allow more precise calculation of the caloric requirements during overfeeding, 24h-EE was measured twice during eucaloric conditions in the baseline period. Energy intake during the first measurement in the eucaloric respiratory chamber was based on unit-specific calculations. The caloric amount of the meals eaten during the second eucaloric measurement was equal to the 24h-EE calculated from the first assessment. For all analyses, the results from the second eucaloric measurement were considered the baseline 24h-EE. The 24h-EE response during fasting and overfeeding (200% of the standard WMD during baseline) was determined. During CR, energy intake during the 24h-EE assessment was the same 50% calorie-reduced liquid diet eaten when not in the calorimeter. Because of the availability of the calorimeter, not every weekly 24h-EE assessment during weight loss was completed by all 12 subjects (24h-EE assessment in week 1 was completed by 11 individuals, by 5 in week 2, by 11 in week 3, by 2 in week 4, by 3 in week 5, and by 12 in week 6).
Bomb Calorimetry and Energy Intake and Waste: To measure accurately the energy content of provided food, urine, and stool, samples were combusted using the Isoperibol Calorimeter 6200 with a model 1108 oxygen bomb; details about this method are described elsewhere (Parr Manual no. 483 M, 6200 Calorimeter Operating Instruction Manual; Parr Instrument Co., Moline, IL). The meals provided during weight-maintenance periods were prepared in a metabolic kitchen using The Food Processor Software (ESHA Research 10.0.0; Salem, OR). On days of bomb calorimetry of the provided foods (Fig. 1), two identical meals were prepared; one meal was selected at random and given to the volunteer and the other meal was combusted to determine actual caloric content. Unconsumed food during the baseline period and the periods after CR was returned to the metabolic kitchen and its caloric content was measured by bomb calorimetry. The resulting calories (<5% of overall calories) were subtracted from those measured for the day's meals.
We used nonabsorbable dye markers (FD&C blue) to determine the exact beginning and end of each 3-day stool transition period. Stool samples were collected from the appearance of the first marker until the appearance of the second marker. Stool samples for calorimetry were stored at −20°C, and after the 3-day collection period, the sample was weighed and distilled water equal to the weight of the stool was added. Samples were subsequently homogenized and followed by lyophilization (freeze-drying) of the feces–water slurry. Urine was collected from administration of the initial dye marker through administration of the second marker (i.e., for three consecutive 24-h periods). Daily urine collections underwent direct lyophilization, which was performed at −77°C with a Freezemobile 12XL instrument (Virtis, Gardiner, NY). After completion of the drying process, the sample was weighed and ~1-g pellets of dried feces or urine were produced with a pellet press (Parr Instrument Co.). Thereafter, the energy content of stool and urine samples was analyzed using bomb calorimetry.
Activity Level. Physical activity was measured at all times during CR, including days spent in the calorimeter, using five omnidirectional accelerometers (Actical; Philips Respironics, Bend, OR) that were attached to each volunteer's wrists, ankles, and waist, as previously described. Because of technical difficulties, complete activity-level data from all 42 days of CR was available from only 11 volunteers.
Using the Actical data, a daily activity factor was determined for each subject. First, the daily activity counts of all five activity monitors were combined into a single measure, that is, weighted average sedentary time (AST), using the FFM values of the related body parts from the DXA closest in time to the day in question as weighting factors in the following equation:
This weighted AST then was used to calculate a daily activity factor expressed as the ratio of daily active time to the active time within the chamber during the closest energy expenditure assessment:
Calculations. To investigate the accuracy of measurements of energy intake and expenditure, we calculated the daily energy deficit during CR:
Adjusted daily energy intake was calculated as directly measured food calories minus measured calories excreted in urine and stool. The 24h-EE values from the corresponding weekly respiratory chamber measurement were used. In the case of missing 24h-EE assessment values, the values from the closest respiratory chamber measurement were used. The estimated 24h-EE for days without a measured assessment of 24h-EE was multiplied by the daily activity factor to account for differences in activity while within the respiratory chamber versus while on the clinical research unit.
Each individual's assumed total-body energy deficit resulting from the 6 weeks of CR was estimated from the absolute change in body mass (ΔFM and ΔFFM in kilograms) using the DXA values from the baseline period and the last week of CR. The calories were extrapolated to 42 days. We applied previously published values of energy contents of FM (9,293.5 kcal/kg) and FFM (1,019.9 kcal/kg):
Finally, we calculated the difference between the sum of the daily calculated energy deficit and the assumed energy deficit (based on the ΔFM and ΔFFM) after 6 weeks of CR.
The energy deficit required to lose 1 kg of body weight was calculated by dividing the calculated energy deficits by the absolute weight changes.
Statistical analysis was performed using the SAS statistical software package (SAS E-guide 4.2 and SAS version 9.2; SAS Institute, Cary, NC). Unless otherwise specified, data are expressed as means ± SDs. Pearson correlation coefficients were used to examine associations between variables, unless otherwise specified. Sex differences were assessed using the Student t test. Anthropometric and metabolic characteristics before and after CR were compared using paired t tests. The impact of baseline predictors on weight change over 42 days was assessed in a mixed model[18,19] to account for both repeated measures using a first-order autoregressive covariance structure and the quadratic relationship of weight change with time. Race, baseline weight, sex, and age were included as covariates in all mixed models. Similar models were used to understand predictors of the accumulation of the calculated energy deficit but using a linear relationship with time. For illustrative purposes, we categorized spendthrift and thrifty subjects as those with 24h-EE responses to fasting above and below the median value, respectively, and differences between groups were compared using the Student t test. For analyses involving weight changes, we compared weights measured at the end of the baseline metabolic stabilization period to weights on the last day of CR. All reported weight and 24h-EE changes are expressed as percentage changes from the baseline values to account for differences in baseline body size between subjects.
Diabetes. 2015;64(8):2859-2867. © 2015 American Diabetes Association, Inc.