Acetoacetates

This study had a single arm before-and-after comparison design. The experimental intervention consisted of a KD without caloric restriction lasting 6 weeks (42 days) with a previous preparation period including detailed instructions during classes and individual counselling by a dietitian. Day one and day 42 will subsequently be denoted PRE and POST, respectively.
Our dietary recommendations and handouts were similar to those used in the study by Klement et al. [14 ]. Our subjects were provided with handouts summarizing the main aspects of a KD and given a list of suitable foods with very low carbohydrate content. Furthermore, the subjects shared cooking recipes and links to helpful webpages on an internal weblog. They were free to follow a KD according to their personal preferences but were advised to eat ad libitum but limit their carbohydrate intake to a maximum of 20–40 g/day to derive at least 75%, 15–20%, and 5–10% of total energy from fats, protein, and carbohydrates, respectively. In the first intervention week the subjects were instructed to switch in a gradual and well-controlled manner from their usual to a KD with the objective to attain stable ketosis by the end of the week, as such transition periods can be accompanied by short-term side effects including gastrointestinal symptoms (eg, constipation) and slight headache [15 ]. The subjects received a logbook to record daily any side effects and complaints during the KD intervention. To avoid biasing the cardiopulmonary exercise testing at POST, the subjects were advised not to alter their physical activities during the study period. Physical activity was assessed at PRE and POST using a validated questionnaire developed by Frey et al. [16 (link)].
Compliance with the dietary regimen was monitored by taking daily measurements of urinary ketones and keeping 7-day food records. The subjects documented their daily urinary ketone measurements (acetoacetate) using self-testing strips (Ketostix, Bayer Vital GmbH, Leverkusen, Germany). An initial substudy revealed that ketonuria can be most reliably detected in the early morning and post-dinner urine [17 (link)]. Those results also enabled our dietitian to individually fine-tune their diets if necessary via phone or personal contact, thus ensuring continuous ketosis.
Two semi-quantitative 7-day food records were obtained from all subjects before and during the last week of the intervention. Our dietitian gave them precise oral and written instructions individually on how to accurately record the amounts and types of food and beverages. Subjects were given a digital portable scale (KS 22, Beurer GmbH, Ulm, Germany) and instructed to weigh all food items separately if possible or to estimate the amounts and take a photograph. The energy, macro- and micronutrient intakes were analysed with a nutritional database software (Prodi 6.5 basis, Nutri-Science GmbH, Stuttgart, Germany).

The primary efficacy end-point was the total daily dose of insulin required to achieve euglycemia, which was defined as a daily mean pre-prandial blood glucose concentration of ≤ 7.8 mmol/L (140 mg/dL). The secondary efficacy end-points including EE and RQ, as measured by indirect calorimetry performed one day before treatment day, day 7 and after 12 weeks. Oxygen consumption and carbon dioxide production were measured to calculate the EE and RQ. According to the Weir equation, EE was calculated without using urinary urea nitrogen levels [14 (link)]. Protein oxidation was determined from 24-h urinary nitrogen excretion, and the carbohydrate and lipid oxidation rates were determined from the non-protein RQ. EE and RQ were assessed at the steady state, which was defined as the 10-min time period during which the average minute-by-minute changes in oxygen consumption and carbon dioxide production were < 10% and the average RQ change was < 5% [16 (link)]. The average RQ and EE at the steady state were measured at each 10-min recording period.
The exploratory end-points included the changes in daily urinary glucose excretion, 3β-hydroxybutyrate, acetoacetate concentrations, body weight, systolic blood pressure, and diastolic blood pressure.