The study population was initially composed of 24 T2DM patients. Two patients, due to personal reasons (completely unrelated to the study), were unable to participate in the second phase of the project, and a further patient complained of self-diagnosed wheat-related complications during the first phase (notwithstanding the written consent provided at the outset). These three patients were, therefore, excluded from the study. The final study population was comprised of 21 T2DM patients (14 men; 7 women). The mean age was 64.4 ± 10.9 with a mean body mass index (BMI) of 27.9 ± 4.7 (Table 1). All patients were recruited on entry to the clinic following consultation at the Unit of Clinical Nutrition of the Department of Experimental and Clinical Medicine, University of Florence, Careggi University Hospital.
T2DM was confirmed if at least one or more of the following were reported: (a) HbAc1 ≥6.5 %, (b) fasting plasma glucose ≥126 mg/dL (7.0 mmol/L), (c) 2-h plasma glucose ≥200 mg/dL (11.1 mmol/L) during an oral glucose tolerance test, and (d) a random plasma glucose ≥200 mg/dL (11.1 mmol/L) in patients with symptoms of hyperglycemia. The above-mentioned criteria were consistent with current criteria for the diagnosis of T2DM, according to the American Diabetes Association (2014). Exclusion criteria included clinical manifestations of microvascular complications (retinopathy, neuropathy and nephropathy), macrovascular complications (cardiovascular disease), inflammatory bowel disease, and excess alcohol intake, celiac disease, gluten-sensitivity and wheat allergies.
Patients were instructed not to alter their dietary or lifestyle habits, and written informed consent was then obtained from each participant before the start of the trial. The institutional review board at the University of Florence approved the study protocol.
Data collection and measurements
Patients underwent an interview, according to standardized methods, to obtain information about personal medical history, demographics, medication, and lifestyle habits. All information relating to the above-mentioned aspects served as descriptive supplementary information pertaining to the current study population, but was not used as a basis for patient exclusion.
Physicians, using standardized protocols, conducted a physical examination, blood pressure measurements, laboratory tests and a dietary survey. BMI was calculated as weight (kg)/height (m2). Participants that were smoking at the time of the physical examination were registered as smokers. If physical activity over the preceding 6 months did not meet certain criteria (based on duration and intensity), patients were then documented as having a sedentary lifestyle. Hypertension (raised blood pressure) was defined as systolic blood pressure 140 mmHg or more and/or diastolic blood pressure 90 mmHg, in accordance with the guidelines of the European Society of Cardiology. Dyslipidemia was defined according to the Third Report of the National Cholesterol Education Program (NCEP-III), or if patients reported taking anti-dyslipidemic drugs, as verified by the physician. Adherence to a Mediterranean diet was evaluated from a questionnaire that included 3 categories of consumption (min 0 points, max 2 points) for each food group (cereals, fruit, vegetables, legumes, olive oil, meat products, dairy products, fish and alcohol) representing the major constituents of the Mediterranean diet. The questionnaire permitted the assignment of an overall score (min 0 points, max 18 points) to each patient, thereby ranking the degree of adherence .
Experimental and control wheat
The ancient experimental wheat utilized in the present study was organic KAMUT® khorasan wheat (Tritucum turgidum subsp. turanicum), provided by Kamut Enterprises of Europe (KEE), Belgium (from here on referred to as the khorasan wheat). KAMUT® is a registered trademark of Kamut International, Ltd. and Kamut Enterprises of Europe, bvba and guarantees the wheat is pure ancient khorasan wheat and is organically grown and processed. As the control (from here on referred to as the control wheat), a mix of organic modern commercial Italian durum (T. durum) varieties and soft wheat (T. aestivum) varieties, respectively, were used. Various reported biochemical parameters of the khorasan and control semolina and flour (Table 2) were determined as previously . Given that different countries utilize different terminology to classify the different types of milled flour, we report the ash content (positively correlated to the extraction rate) for comparative purposes. Khorasan semolina (ash content 1.10–1.35 %) and flour (ash content 1.0 %) were processed by Molino SIMA S.C.A.R.L. (Argenta, Ferrara, Italy). For the organic khorasan wheat, different milling procedures were employed to produce the granulated semolina (analogous to semi-whole wheat semolina) and flour (analogous to semi-whole wheat flour), respectively, thereby resulting in differences in ash content. For the control, semi-whole-wheat granulated semolina (ash content 1.0–1.35 %) and semi-whole wheat flour (ash content 0.95 %), respectively, were similarly processed by Molino SIMA.
Pastificio Artigiano FABBRI s.a.s. (Strada in Chianti, Firenze, Italy) prepared the pasta (with no additives) from both the khorasan and control semolina, according to the artisan manufacturing procedures. The artisan enterprise of Panificio Menchetti Pietro di Santi e Figli s.n.c. (Cesa Marciano della Chiana, Arezzo, Italy) prepared the bread, biscuits and crackers using the khorasan and control flour. Naturally, leavened Tuscan-style sourdough bread was prepared. Besides the flour composition, dry crackers contained 20 % extra-virgin olive oil. The biscuits were prepared using 10 % sugar, 5 % butter, and one egg per 100 g flour.
The study was a randomized, double-blinded, crossover trial aimed at testing whether a replacement diet with khorasan wheat products and/or control wheat products could provide additive benefits to T2DM patients. For this reason, other cereals were excluded from the diet and “replaced” by either the khorasan or control products during the respective intervention phases. The food products were packaged with no labels attached to the packages, and patients were informed that all products to be administered were organic and prepared by artisan methods. The patients were randomly divided into two groups (Group A and Group B), and a crossover study design with two intervention phases was implemented. The first intervention phase was initiated in late January 2015, with Group A and B, respectively, initiating the trial with organic khorasan and control products. Participants in both groups received 500 g/week of pasta, 150 g/day of bread, 250 g/week of crackers, and 250 g/week of biscuits for a period of 8 weeks. Patients were advised to eat the products according to their normal cereal consumption habits, which were documented at baseline. A washout period of 8 weeks was then affected, during which patients were permitted to eat all foods according to their “normal” dietary habits. The second intervention phase of 8 weeks was initiated in early May 2015, and Group A and B crossed-over to consume the control and khorasan products, respectively. On average, patient’s daily intake of both khorasan and control semolina was 62 g dry weight, whereas daily intake of khorasan or control flour (from all the products consumed) amounted to 140 g dry weight. The energy (kcal) provided by semolina and flour was 221 and 501 kcal, respectively, making a total of 722 kcal (50–55 % of daily energy intake) replaced in this intervention. At baseline and after each intervention, all subjects were examined between 7:00 a.m. and 9:30 a.m. after an overnight fasting period. Furthermore, subjects were asked not to engage in strenuous physical activity during the day before the examination.
Venous blood samples were collected into evacuated plastic tubes (Vacutainer). Samples were centrifuged at 3000g for 15 min (4 °C) and stored in aliquots at −80 °C until further analysis. Cholesterol subtypes, triglycerides, glucose, insulin, HbA1c, and serum electrolytes were assessed according to conventional methods. Pro- and anti-inflammatory cytokines were determined by using the Bio-Plex cytokine assay (Bio-Rad Laboratories Inc., Hercules, CA, USA), according to the manufacturer’s instructions.
Assessment of ROS production and total antioxidant capacity
Leukocyte (lymphocyte, monocyte and granulocyte) ROS generation was measured as reported previously [8, 9]. Similarly, fatty acid peroxidation was determined by measuring malondialdehyde (MDA) using the thiobarbituric acid reactive substance (TBARS) assay kit (Oxitek-ZeptoMetrix Corporation Buffalo, NY, USA). Total antioxidant capacity (TAC), accounting for total hydrophilic ROS scavengers, was measured using the ORAC assay (oxygen radical absorbance capacity), based on the inhibition of the peroxyl-radical-induced oxidation initiated by thermal decomposition of azo-compounds, like 2,2-azobis (2-amidinopropane) dihydrochloride (AAPH), was performed as previously described .
The statistical package PASW 20.0 for Macintosh (SPSS Inc., Chicago, IL, USA) was utilized. Results were expressed either as mean ± SD or as median and range. One-way ANOVA was used for testing differences between khorasan and control flour and semolina. The analyses were simplified by calculating the absolute change for each variable tested (mean value at baseline subtracted from the mean value after intervention for each subject) with independent t sample tests. All data were treated as paired samples from a crossover study. Data that were not normally distributed were logarithmically transformed. The two interventions were analyzed by taking into account both phases in the two groups of subjects at different stages. Data were analyzed by using paired t tests for significant differences between changes observed during test and control intervention periods. Moreover, in order to compare the effect of organic khorasan products versus baseline and versus the control products, a general linear model for repeated measurements, after adjustment for age and gender, modifiable risk factors, diet quality, and antidiabetic medication was performed. A value of p ≤ 0.05 was considered to indicate statistical significance.