Ketostix

All organ systems were clinically examined according to Dirksen [39 ] at day -5 and day 4. The cows were not considered healthy in presence of: urine ketone ≥ ++ Ketostix, (Bayer AG, Leverkusen, Germany), rectal temperature (RT) > 39.5°C, lesions proximal to the feet causing lameness, lameness located in the front limbs, swollen and painful udder, purulent vaginal discharge, gastrointestinal disorders, cardiac murmurs, severe infection of respiratory tract or nervous disorders. If any cow was considered not to be healthy during any of the research periods, it was excluded from the experiment.
BW was estimated using a measuring tape according to Yan et al. [40 (link)]. To estimate body condition, Edmondson’s BCS [41 (link)] was determined. WH was measured at the level of the forelimb, using a meter ruler with a horizontal rod (including a spirit level). For BCS, BW and WH, the means of the 2 measurements were taken for further analysis. As BCS is recorded on a quarter point scale, the mean value was rounded up to the next quarter.

Pathology of the GBM was confirmed by neuropathology review. Patients with recurrent GBM had to have MRI-documented tumor progression or recurrence. Baseline laboratory studies included serum electrolytes, renal and liver functions, CBC, PT/PTT, fasting blood glucose (FPG), and serum lipid profile (cholesterol, triglycerides, high-, low-density lipoprotein, [HDL, LDL]), uric acid levels, and serum BHB.The subject’s known food allergies and special (e.g., religious) dietary requirements were reviewed. Participants were taught to measure urine ketone body (KB) levels using Ketostix (Bayer AG, Germany) which measures acetoacetate, and blood for glucose and ketone levels using self-administered Precision Xtra® Meter (Abbot Diabetes Care, Alameda, CA, USA) which measures BHB. These were done fasted in the morning and 2 h post-prandially in the evening. Subjects were instructed to keep urine ketone/blood glucose and ketone diary.

In order to increase motivation and adherence, study participants were female dietetics students with both knowledge and interest in dietary surveys. Two female dietetic students were involved in the planning of the two diets, and great importance was given to composing menus that could be attractive to the participants in the study. At baseline assessments, the research nurse administered a daily diet and exercise logbook. Participants were instructed to note on a daily basis any deviations from the dietary protocol and to note morning weight after first void. Participants were instructed to eat extra pre-prepared snacks if losing weight. Since a ketogenic diet should lead the body to enter a state of increased fat burning and induce ketosis, participants were also instructed to have their morning urine dipped for ketones daily using a standard nitroprusside-based dipstick test (Ketostix, Bayer, Basel, Switzerland). They were instructed to read the color change after 15 seconds and to note the test result in their daily diet and exercise logbook.

Due to the limitations imposed by the ambulatory setting in our clinic, all KDs were self-administered with counselling at least once per week. Patients received short handouts providing basic information on KDs such as main foods to eat and avoid as well as sample menus. For more detailed information we also lent and recommended to buy a popular book on KDs for cancer patients [15 ]. In general we recommended to restrict CHO intake to <50 g/day and emphasized the intake of olive oil, coconut oil, butter, ghee, and fatty fish, cheese, meat and non-starchy vegetables. To monitor ketosis, patients were provided with urine strips (Ketostix^®, Bayer Consumer Care AG, Basel, Switzerland) and a diary for protocolling their measurements, preferably done in the afternoon. Patients were also asked to record the quantity of all foods eaten for at least 2 days in order to determine their actual energy and macronutrient intake.

In order to evaluate dietary adherence, participants were asked to make a daily note of any deviations from the dietary protocol. A previous publication describes the results regarding dietary intake, calculated from participants’ daily notes on deviations from planned food, and levels of ketone bodies in urine and blood [27 (link)]. The results showed excellent compliance to the planned diets and urinary ketosis was detectable after a maximum of four days and thereafter throughout the LCHF diet intervention (Ketostix, Bayer, Basel, Switzerland) (Supplemental Figure S1), and all women had blood β-hydroxybutyrate concentrations >0.5 mM at the end of the LCHF diet intervention. Participants also maintained their habitual physical activity levels [27 (link)].
During the intervention, weight was reduced during both the LCHF (~3 kg) and the control (~1 kg) diet [27 (link)].