This study was approved by the Ethic Committee of Institute of Food Technology and Nutrition research, Shahid Beheshti University of Medical Sciences and was conducted in 2008 at Research Institute for Endocrine Sciences (RIES), Tehran, Iran.
Sixty-three participants with type2 diabetes whose diabetes was controlled by metformin were recruited and 56 completed the trial. At the beginning, the protocol and the aim of study were fully explained to the participants and written informed consent was obtained from each volunteers. Inclusion criteria, including diagnosed type2 diabetes mellitus for more than 5Y, diagnosis after 30 years old, age of 35 to 50Y, body mass index (BMI; in kg/m2) >25 and <30 and fasting blood glucose of 126–180 mg/dl. Exclusion criteria were the history of myocardial infarction, angina pectina and stroke, diagnosis of cardiovascular disease, liver or renal disease or chronic inflammatory and thyroid disease, being vegetarians or vegans, smoking, consuming of alcohol and any supplements (e.g. vitamins such as C and E, fish oils, CLA, etc.) 2 months before intervention, pregnancy and menopause.
The study was an eight weeks randomized double blind, placebo-controlled (RCT) parallel intervention. The participants were stratified according to their sex, age and BMI into one of three groups (A, B, and C) receiving 3.0 g CLA/d (3×1 g capsules; a 50:50 isomer blend of c-9, t-11 and t-10, c-12 CLA) with 100 IU/d VitE, 3.0 g CLA/d with VitE placebo, or CLA placebo (soy bean oil) and VitE placebo respectively. Ineffectiveness of soybean oil as a placebo in the quantities being used in this study on reviewed variables has shown in other studies . The CLA used in this study was Tonalin SG1000T FFA and soft gelatin capsules with clear, transparent shell and pale amber fill. They contain Tonalin FFA 80 i.e., free fatty acids containing about 80% conjugated Linoleic. All supplements were supplied by Cognis, Norway. Each volunteers received the capsules in 2 batches, at the beginning of the study. All participants were asked to maintain their usual physical activity and dietary and lifestyle habits and these were checked by food record and physical activity questionnaire. There was no change in prescribed medication throughout the trial.
Acceptance of the supplements was investigated via weekly phone calling and meetings through asking and counting the remaining capsules in the package delivered. During these calls possible problems such as supplement intolerance and medication use, possibly changes in food consumption, getting a new disease or a change in physical activity was followed and if this situation occurred, the patient desired were excluded. In the fourth and eighth weeks of study through counting the remaining capsules, compliance rates of patients was evaluated and patients were not consumed more than ten percent of received capsules, were excluded from the study.
To evaluate the mean dietary intake two 1-d 24-h food recall and 2-d food records (one day a week holiday) were used on the baseline and the end of the fourth and eighth week of the study. In the first taking the 24 days recall, how to diet properly recording, including how to weigh and measure food was trained by an expert. This dietary information was analyzed with N4 software (Nutritionist: version 4.0; Tinuviel Software, Warrington, United Kingdom).
Body weight was measured with light clothing but no shoes on a digital balance (with 0.1 kg sensitivity). Height was assessed by using a stadiometer that measured to the nearest 0.1 cm. Body mass index (BMI) was estimated as the ratio of body weight to height squared and expressed as kg/m2. Waist circumference (with 0.1 cm sensitivity) was measured at the minimum circumference between the iliac crest and the last rib cage at the end of exhalation. The hip circumference was measured using tape as the maximal circumference over the hip and Waist-to-hip ratio (WHR) was calculated . Bioelectrical impedance analysis was used to measurement of body fat and lean percentage (Body Stat 1500, Douglas Isle of man, British islets, England). For this purpose, first two glasses of water consumed by patients and after one hour and urination without any metal on the body, body composition was measured. Measuring body composition was done in lying state and installing the electrodes to the right hand and foot. Patients were taught avoiding intense activity the day before testing . These measurements were done at the beginning and end of the study.
Blood pressure measuring was done to the nearest 2 mmHg, after resting for at least 15min and sitting on the seat handle. Right arm blood pressure was measured, twice, at least five minutes interval, by Korotkoff’s auscultatory method at the beginning and end of the study.
Seven ml 12-hour fasting state and 3 ml postprandial brachial vein blood samples were taken at the baseline and end of eighth week and blood collected into EDAT containing tubes. A standard breakfast in diabetic patients is contained about 360kcal, including: 56.6 g carbohydrate (50-55% Cal), 19.5g protein (15-20% Cal) and 11.5 g fat (30% Cal) [24, 25]. Localized standard breakfast for Iranians that has been used in previous studies included two servings bread; one serving cheese, 2x dates, and four small cubes that total provided about 390kcal and 14g protein . To maintain comparability of Iranian studies with the international studies in which the breakfast induced energy is 360 kcal, and also given that patients participating in this study did not normally use refined s such as in the diet, of breakfast was removed and replaced as a number of dates were added to the breakfast.
Serum glucose concentration was measured by using enzymatic colorimetric method according glucose oxidase principle (Glucose determination kit, Parsazmun, Tehran, Iran) through auto-analyzer instrument (Sellectra II, Dieren, Netherland). Serum insulin, proinsulin and C-peptide levels were measured by Enzyme Linked Immuno assay (ELISA) kit (Mercodia AB, Uppsala, Sweden). Glycated hemoglobin was determined on whole blood sample by ion exchange chromatography method (HbA1c Kit, Inter Medical, Villaricca, Italy).
The intra assay coefficient of variation (CV%) for glucose, insulin, proinsulin, C- peptide and HbA1c were 4.7%, 5.5%, 4.5%, 4.7% and 5.6% and the inter assay coefficient of variation were 4.9%, 5.8%, 4.9%, 5%, and 5.8% respectively. The assays sensitivity was 1mg/dl, 1mU/L, 0.5 pmol/l, 5 pmol/l and 1% respectively.
Insulin resistance was calculated according the homeostasis model of assessment ratio (HOMA-IR) formula, as an index of insulin resistance:
[Insulin (μU/ml)×glucose (mmol/L)]/22.5 . That 5<HOMA IR is defined as insulin resistance and 3> HOMA IR as not-insulin resistance .
Insulin sensitivity was calculated from following formula [29
And beta cell function determined through proinsulin/ insulin ratio . Beta cell function index as HOMA-B% and C-peptide to insulin molar ratio as an index of liver insulin clearance was calculated .
Fasting b-cell responsiveness (M0) represents the ability of fasting glucose to stimulate b-cell secretion and postprandial b-cell responsiveness (M1) represents the ability of postprandial glucose to step up b-cell secretion and they were calculated using the formula of Hovorka et al. [32
Serum concentration of triglyceride and HDL cholesterol were measured using kits and enzymatic colorimetric method (Parsazmun, Tehran, Iran). Total cholesterol Concentration was measured by enzymatic photometric method kits (Parsazmun, Tehran, Iran). LDL cholesterol concentration calculated using the Friedewald formula. Also, the ratio of LDL to HDL cholesterol concentrations was calculated. The intra assay coefficient of variation (CV%) for triglycerides, total cholesterol and HDL cholesterol were 2.7%, 2.3% and 5.1% and the inter assay coefficient of variation were 2.9%, 2.5T and 5.5% respectively. Also, the related assays sensitivity was 1 mg/dL, 3mg/dL and 1mg/dl respectively.
Serum leptin levels were measured by Enzyme Linked Immuno assay (ELISA) kit (Diagnostics Biochem Canada Inc., ontario, Canada). Serum adiponectin levels were measured by Enzyme Linked Immuno assay (ELISA) kit (Mercodia AB, Uppsala, Sweden). The intra assay coefficient of variation (CV%) for leptin and adiponectin were 2% and 1.5% respectively and the inter assay coefficient of variation were 4% and 2%. The sensitivity of the assays was 0.50 ng/ml and 1.25 ng/ml respectively.
Serum concentrations of interleukin-1 beta, interleukin-6 and TNF-α were measured using ELISA kits (Diaclone, France) and CRP concentration using ELISA kit (Diagnostics Biochem Canada Inc., Ontario, Canada). Intra assay coefficient of variation of inflammatory IL-1 beta, interleukin 6, CRP and TNF-α, were 7.1%, 4.6%, 1.8%, 3% and the related inter assay coefficient of variation were 7%, 4.7%, 2% and 3.2% respectively. Sensitivity of assays were also 7 pg/ml, 2 pg/ml, 10 ng/ml and 8 pg/ml. Measuring fibrinogen concentration and PAI-1was done using ELISA kits (Hyphen BioMed, Neuville-Sur-Oise, France) and MDA concentration by using the colorimetric method kit (Cayman Chemical Company, Ann Arbor, USA). The intra and inter assay coefficient of variation for fibrinogen and PAI-1, were 2.6%, 3.2% and 3%, 3.5% respectively and assays sensitivity were 1 mg/ml and 0.5 ng/ml respectively. These values for apoB-100 and MDA were 1.4%, 3% and 2%, 3.5% and 0.1 μg/dl, 1 μmol/L respectively.
All statistical tests were performed with the use of SPSS (version 13.0; SPSS Inc, Chicago) and a p-value<0.05 showed statistical significance. Normality and homogeneity of variance was tested with Kolmogrov-Smirinov. ANOVA test were used to compare mean differences before and after intervention for anthropometric data and body composition. Other changes were compared using ANCOVA test adjusted to body composition and waist circumference. To compare food consumption at the baseline and end of the fourth and eighth weeks among groups, and comparison of food consumption in each group among baseline and end of the fourth and eighth weeks of study the ANOVA test and repeated measure ANOVA test were used.