Study population

This is a follow up study on a published clinical trial [6]. It was an open, uncontrolled, nutritional intervention clinical trial conducted for 4 months, and performed in a single center.

The patients attending the Obesity Unit at the Complejo Hospitalario Universitario of Santiago de Compostela, Spain to receive treatment for obesity were consecutively invited to participate in this study.

The inclusion criteria were, age 18 to 65 years, body mass index (BMI) ≥30 kg/m², stable body weight in the previous 3 months, desire to lose weight, and a history of failed dietary efforts. The main exclusion criteria were, diabetes mellitus, obesity induced by other endocrine disorders or by drugs, and participation in any active weight loss program in the previous 3 months. In addition, those patients with previous bariatric surgery, known or suspected abuse of narcotics or alcohol, severe depression or any other psychiatric disease, severe hepatic insufficiency, any type of renal insufficiency or gouts episodes, nephrolithiasis, neoplasia, previous events of cardiovascular or cerebrovascular disease, uncontrolled hypertension, orthostatic hypotension, and hydroelectrolytic or electrocardiographic alterations, were excluded. Females who were pregnant, breast-feeding, or intending to become pregnant, and those with child-bearing potential and not using adequate contraceptive methods, were also excluded. Apart from obesity and metabolic syndrome, participants were generally healthy individuals.

The study protocol was in accordance with the Declaration of Helsinki and was approved by the Ethics Committee for Clinical Research of Galicia, Santiago de Compostela, Spain (registry 2010/119). Participants gave informed consent before any intervention related to the study. Participants received no monetary incentive.

Nutritional intervention

All the patients followed a VLCK diet according to a commercial weight loss program (PNK method®), which includes lifestyle and behavioral modification support. The intervention included an evaluation by the specialist physician conducting the study, an assessment by an expert dietician, and exercise recommendations. This method is based on a high-biological-value protein preparations obtained from cow milk, soya, avian eggs, green peas and cereals. Each protein preparation contained 15 g protein, 4 g carbohydrates, 3 g fat, and 50 mg docohexaenoic acid, and provided 90–100 kcal.

The weight loss program has five steps (Additional file 1: Figure S1) and adheres to the most recent guidelines of 2015 European Food Safety Authority (EFSA) on total carbohydrates intake [3]. The first three steps consist of a VLCK diet (600–800 kcal/day), low in carbohydrates (< 50 g daily from vegetables) and lipids (only 10 g of olive oil per day). The amount of high-biological-value proteins ranged between 0.8 and 1.2 g per each kg of ideal body weight, to ensure patients were meeting their minimal body requirements and to prevent the loss of lean mass. In step 1, the patients ate high-biological-value protein preparations five times a day, and vegetables with low glycemic indexes. In step 2, one of the protein servings was substituted by a natural protein (e.g., meat or fish) either at lunch or at dinner. In step 3, a second serving of low fat natural protein was substituted for the second serving of biological protein preparation. Throughout these ketogenic phases, supplements of vitamins and minerals supplements, such as K, Na, Mg, Ca, and omega-3 fatty acids, were provided in accordance to international recommendations [22]. These three steps were maintained until the patient lost the target amount of weight, ideally 80%. Hence, the ketogenic steps were variable in time depending on the individual and the weight loss target.

In steps 4 and 5, the ketogenic phases were ended by the physician in charge of the patient based on the amount of weight lost, and the patient started a low-calorie diet (800–1500 kcal/day). At this point, the patients underwent a progressive incorporation of different food groups and participated in a program of alimentary re-education to guarantee the long-term maintenance of the weight loss. The maintenance diet, consisted of an eating plan balanced in carbohydrates, protein, and fat. Depending on the individual the calories consumed ranged between 1500 and 2000 kcal/day, and the target was to maintain the weight lost and promote healthy life styles.

During this study, the patients followed the different steps of the method until they reach the target weight or up to a maximum of 4 months of follow-up, although patients remained under medical supervision for the following months.

Schedule of visits

Throughout the study, the patients completed a maximum of 10 visits with the research team (every 15 ± 2 days), of which four were for a complete (C) physical, anthropometric and biochemical assessment, and the remaining visits were to control adherence and evaluation of potential side effects. The four complete visits were made according to the evolution of each patient through the steps of ketosis as follows: visit C-1 (baseline), normal level of ketone bodies; visit C-2, maximum ketosis (approximately 1–2 months of treatment); visit C-3, reduction of ketosis because of partial reintroduction of normal nutrition (2–3 months); visit C-4 at 4 months, no ketosis (Additional file 1: Figure S1 and Fig. 1a). The total ketosis state lasted for 60–90 days only. In all the visits, patients received dietary instructions, individual supportive counsel, and encouragement to exercise on a regular basis using a formal exercise program. Additionally, a program of telephone reinforcement calls was instituted, and a phone number was provided to all participants to address any concern.

Anthropometric assessment

All anthropometric measurements were undertaken after an overnight fast (8 to 10 h), under resting conditions, in duplicate, and performed by well-trained health workers. Participant’s body weights were measured to the nearest 0.1 kg on the same calibrated electronic device (Seca 220 scale, Medical Resources, EPI Inc. OH, USA), in underwear and without shoes. BMI was calculated by dividing body weight in kilograms by the square of height in meters (BMI = weight (kg)/height² (m).

Resting metabolic rate

The RMR was measured by indirect calorimetry using a portable desktop metabolic system (FitMate PRO, Cosmed, Rome, Italy) and under overnight fasting conditions. Participants were instructed to arrive at the hospital by car, to minimize vigorous physical activity during the 24 h prior to the measurement, and to avoid drinking caffeinated beverages for at least 12 h before testing. All participants rested supine for at least 20 min. During this resting time, the body composition (bone mineral density, lean body mass and fat mass) was determined, and then rested in sitting position in a quiet and darkened room for a further 15 min before the test.

Test-re-test validation was performed and after resting, oxygen consumption was measured continuously for 15 min under thermo-neutral conditions, and the final 10 min of data were used to calculate RMR. The FitMate uses a turbine flow meter for measuring ventilation and a galvanic fuel cell oxygen sensor for determining the fraction of oxygen in expired gases. Moreover, it has sensors for the measurement of temperature, humidity, and barometric pressure for use in internal calculations. The FitMate uses standard metabolic formulas to estimate oxygen consumption, and RMR is calculated using a predetermined respiratory quotient (RQ) of 0.85. During the measurement period, participants remained sitting, breathed normally, and were instructed to remain awake, and to avoid talking, fidgeting and hyperventilating. The reliability of measuring RMR with Cosmed’s FitMate metabolic system have been determined in several previous studies [9, 15, 21], and by in house controls (Additional file 2: Figure S2).

For the purposes of this study measured RMR or the crude values provided by the method were obtained and expected-RMR was defined as the variation in energy expenditure that could be explained by the observed changes in fat-free mass (FFM), because FFM is the main determinant of RMR [13]. Firstly, we determined the basal energy equivalence per kilogram of FFM in our study population. Then, this quotient was multiplied by the amount of change in FFM between the baseline and each subsequent complete visit. Finally, this product was added to the basal measured RMR, and in this way the expected RMR for each complete visit was obtained.

On the other hand, metabolic adaptation has been described as the change in RMR not explained by changes in FFM [8, 19], and is calculated as the difference between RMR measured at each complete visit and the expected RMR for that visit i.e., Metabolic adaptation = RMR measured – RMR expected.

Total body composition

Body composition was first measured by dual-energy X-ray absorptiometry (DXA; GE Healthcare Lunar, Madison, USA).Daily quality control scans were acquired during the study period. No hardware or software changes were made during the course of the trial. Subjects were scanned using standard imaging and positioning protocols, while wearing only light clothing. For this study, the values of bone mineral density, lean body mass and FM that were directly measured by the GE Lunar Body Composition Software option. Some derivative values, such as bone mineral content, regional lean mass, FFM, and fat mass percentage (FM%), were also calculated.

Multifrequency bioelectrical impedance

Multifrequency bioelectrical impedance (MF–BiA) was also used for determining body composition. FM, FM%, FFM, total body water, intra- and extracellular water, and skeletal muscle mass, were calculated with In Body 720 (In Body 720, Biospace Inc.,Tokyo, Japan). This technology is non-invasive and uses eight contact electrodes, which are positioned on the palm and thumb of each hand and on the front part of the feet and on the heels.

Multifrequency bioelectrical impedance uses the body’s electrical properties and the opposition to the flow of an electric current by different body tissues. The analyzer measures resistance at specific frequencies (1, 5, 50, 250, 500 and 1000 kHz) and reactance at specific frequencies (5, 50, and 250 kHz). The participants were examined lightly dressed, and the examination took less than 2 min and required only a standing position. The validity of this technology has been documented in previous studies [6].

Determination of levels of ketone bodies

Ketosis was determined by measuring ketone bodies, specifically β-hydroxy-butyrate (β-OHB), in capillary blood by using a portable meter (GlucoMen LX Sensor, A. Menarini Diagnostics, Neuss, Germany). As with anthropometric assessments, all the determinations of capillary ketonemia were made after an overnight fast of 8 to 10 h. These measurements were performed daily by each patient during the entire VLCK diet, and the corresponding values were reviewed on the machine memory by the research team in order to control adherence. Additionally, β-OHB levels were determined at each visit by the physician in charge of the patient. The measurements reported as “low value” (< 0.2 mmol/l) by the meter were assumed as to be zero for the purposes of statistical analyses.

Biochemical parameters

During the study all the patients were strictly monitored with a wide range of biochemical analyses. However, for the purposes of this work only certain values are reported. Serum tests for total proteins, albumin, prealbumin, retinol-binding protein, red cell and white cells counts, uric acid, urea, creatinine and urine urea were performed using an automated chemistry analyzer (Dimension EXL with LM Integrated Chemistry System, Siemens Medical Solutions Inc., USA).Thyroid-stimulating hormone (TSH), free thyroxine (FT4), and free triiodothyronine (FT3) were measured by chemiluminescence using ADVIA Centaur (Bayer Diagnostics, Tarrytown, NY, USA). All the biochemical parameters were measured at the 4 complete visits.

The overnight fasting plasma levels of leptin were measured using commercially available ELISA kits (Millipore, MA, USA). The fasting plasma levels of fractionated catecholamines (dopamine, adrenaline and noradrenaline) were tested by high pressure liquid chromatography (HPLC; Reference Laboratory, Barcelona, Spain).

Statistical analysis

The data are presented as means (standard deviation). Each subject acted as his own control (baseline visit). The sample size of the current trial was calculated taking the weight loss after treatment (main variable) into account. It was calculated for an effect size ≥15 kg, and a α = 0.05, and a power (1-β) of 90%. Thus, the sample size was established at a minimum of 19 volunteers who finished the nutritional treatment. The sample size provided sufficient power to test for effects on a number of other metabolic variables of interest.

All statistical analyses were carried out using Stata statistical software, release 12.0 (Stata Corporation, College Station, TX, USA). A p < 0.05 was considered statistically significant. Changes in the different variables of interest from the baseline and throughout the study visits were analyzed following a repeated measures design. A repeated measures analysis of variance (ANOVA) test was used to evaluate differences between different measurement times, followed by post hoc analysis with Tukey’s adjustment for multiple comparisons. In addition, multivariate linear regression models were fitted to assess the potential predictive factors of RMR at each complete visit. The regression models included fat-free mass, FT3, catecholamines (i.e. noradrenaline, adrenaline and dopamine), leptin and β-OHB as plausible determinants of RMR.