Subjects

The pilot study group consisted of 106 Rome council employees (19 males and 87 female; mean age 48, 49 ± 10, 33). Inclusion criteria were: BMI ≥ 25, age > 18 years and < 65 years, currently on a carbohydrate rich diet (> 50% energy), [21] desire to lose weight and health status suitable for a modified ketogenic diet (Tisanoreica®) [22] i.e. normal renal function, not pregnant or lactating. After the start of the experimental protocol the subjects who began new exercise programs or pharmaceutical treatments would be excluded. A small rise in transaminase was not considered a condition for exclusion since mild alterations in GOT and GPT values are common in obese individuals. Subjects eligible for the study were invited to the IPA clinic (Istituto di Previdenza ed Assistenza - health services for public sector employees) to attend an orientation session. At the first visit it was explained that during the first three weeks it was necessary to almost totally exclude carbohydrates and a detailed menu containing permitted and non-permitted foods was provided to each participant, along with the components of the ketogenic Mediterranean with phytoextracts (KEMEPHY) diet described below. Anthropometric measures were performed and blood samples were taken from the subjects two-three days before and after they began the diet. Subjects received no monetary compensation for their participation and signed a voluntary consent form before initiating the diet. The ethical and clinical review committee of IPA and the European Nutrition Society approved the study protocol, informed consent form and information material provided to subjects.

Diet

The KEMEPHY (ketogenic Mediterranean with phytoextracts) diet protocol was ketogenic during the first 3 weeks with approximately 34 g of CHO daily, using low carbohydrate high-protein meals and herbal teas [22] (Tisanoreica® by Gianluca Mech SpA, Orgiano VI) (Table 1 and 2).

Table 1 diet composition in KEMEPHY (ketogenic Mediterranean with phytoextracts) diet. Full size table

Table 2 Plant extracts used in KEMEPHY (ketogenic Mediterranean with phytoextracts) diet Full size table

The permitted foods were: cooked or raw green vegetables (200 g/meal), meat, fish and eggs (2 times/day), olive oil 40 g/day. Integration with a dish (PAT® i.e. porzione alimentare tisanoreica = tisanoreica nutritional portion) composed of high quality proteins (equivalent to 18 grams) and virtually zero carbohydrate (but that mimic their taste) was provided for every meal, for a maximum of four PATs per day. During the last three weeks complex carbohydrates were introduced (50-80 g/day), cheese (60 g/day), PAT was reduced from four to two, while the other indications remained unchanged. The distribution of nutrients (proteins, carbohydrates and fats) in terms of percentage of total caloric intake was 36%, 12% and 52%, respectively (weeks 1 to 3) and 31%, 25% and 44% (weeks 4 to 6). During the 6 weeks, the patients in the study group consumed 20 ml of extract A, 20 ml of extract B and 50 ml of extract C. During the first two weeks, before breakfast and lunch, they also consumed 40 ml of extract D (Tables 2 and 3).

Table 3 Main actives ingredients of used phytoextracts, their reported beneficial effects and related references Full size table

Supplements

Subjects also took a daily (1 caplet each morning) multivitamin supplement [23] (containing Magnesium19 mg, Calcium 16 mg, Phosphorus 8 mg, Zinc 4.5 mg, Iron 4.62 mg, Manganese 1 mg, Potassium 0.5 mg, Copper 0.4 mg, Chromium 28.55 μg, Selenium 4 μg, Niacin 10 mg, Beta carotene 1.8 mg, Folic Acid 66 μg, Biotin 30 μg, Vitamin C 19.8 mg, Vitamin E 3.3 mg, Pantothenic Acid 1.98 mg, Vitamin B6 0.66 mg, Vitamin B2 0.53 mg, Vitamin B1 0.426 mg, Vitamin D3 1.65 μg, Vitamin B12 0.33 μg (Multivitaminico Balestra e Mech, Gianluca Mech SpA, Orgiano VI).

Measurements

Subjects were weighed at the same time of day at the start and after 6 weeks of the diet, using the same weighing scales (Digital Scale Joycare®Jc431). Fasting venous blood samples were collected at weeks 0 and 6 for total cholesterol (CHOLt), triacyglicerol (TG) high-density lipoprotein cholesterol (HDLc), low-density lipoprotein cholesterol (LDLc), glucose, blood urea nitrogen (BUN), uricemia, VES, creatinine, ALT, AST, GGT. Blood was collected in EDTA treated vacutainer tubes. To avoid interassay variation all blood samples were stored at -80° and analysed together at the end of the study. A separate sample of blood was clotted and serum analyzed for total cholesterol and triacylglycerols by photometric assay with an intra-assay and interassay CV values of 2% and 4%, respectively.

HDL cholesterol was determined using a homogenous enzyme immunoassay. The intra-assay and interassay CV values were both < 4%. Plasma glucose was determined colorimetrically using glucose oxidase methodology. Plasma urea nitrogen were measured using an enzymatic (urease), colorimetric method. Creatinine was measured colorimetrically using the picric acid assay, and uric acid was determined using a modified Trinder peroxide assay. LDLc fraction was calculated from Friedewald's formula: LDLc = TC - HDLc - (TG/5) [24]. Body composition was assessed using bioelectrical impedance analysis (BIA Akern Bioresearch, Pontassieve, FI, Italy) which is a non-invasive and portable method for the estimation of fluid compartments, fat and fat-free mass in healthy subjects. Bioelectrical impedance analysis was chosen for the analysis of body composition due to its reliability, safety, convenience and non-invasiveness making it optimal for the routine monitoring of body composition during the ketogenic diet [25, 26]. The experimental design is showed in Figure 1.

Figure 1 Experimental design. Full size image

Statistical analysis

We tested the normality and the assumption of homoscedasticity of all parameters at the start of the trial using the Kolmogorov-Sminorv and Shapiro-Wilk tests. To study changes over time and the effects of gender, we added an appropriate interaction analysis. As there were no significant gender interactions, the data of each group were pooled and analyzed together. A Student's t test was used to compare parameters before and after 6 weeks of the KEMEPHY diet using Statistica Software, ver. 8.0 (Tulsa, USA) and the software package GraphPad Prism version 4.00 for Windows, GraphPad Software, San Diego California USA. All data are expressed as mean ± standard deviation.