This multicenter, randomized controlled trial was conducted from September 2007 to December 2010.
Participants were recruited from three hospitals from Bucharest, Romania. The inclusion criteria were: age over 35 years, BMI over 25 kg/m2 diagnosis of chronic hepatitis C (CHC infection was defined by the presence of anti-HCV antibodies for a least 6 months and a positive HCV-viremia).
The exclusion criteria were: patients with other etiology of chronic liver disease, hepatitis B, autoimmune liver disease, hemochromatosis, HIV infection, patients with history of hepatotoxic or steatosis-inducing drug use, currently on interferon treatment or during the last 12 months, patients having an alcohol consumption of more than 20 g/day for women and 30 g/day for men, history of pancreatitis.
The study was conducted in Bucharest, the most important commercial urban setting of Romania, with a population of 2 million and an estimated CHC rate of 3.35% in adults (data as of 2007).
The DIADIPOHEP (Adipocitokynes, link between virus C hepatitis and type 2 diabetes mellitus)) study was approved by the Romanian National Authority for Scientific Research. Written informed consent was obtained from all participants.
Enrollment began in September 2007 and ended in December 2010. Participants were recruited from three hospitals. Eligibility was established through a screening visit that included a physical examination and a review of the patient's medical history. Following completion of baseline assessments, participants were randomized to a normoglucidic low-calorie diet (NGLCD) group, or to a low-fat diet (LFD) group, both with a lifestlye management program.
The primary endpoint was to measure the impact of dietary changes in reduction of insulin resistance as well as hepatic steatosis and fibrosis through nutritional intervention. Secondary endpoints included changes in weight, lipid profile (total cholesterol, LDL-cholesterol, HDL-cholesterol and triglycerides), blood pressure (systolic and diastolic), hepatic profile, and renal function (estimated glomerular filtration rate [eGFR]).
We performed anthropometric measurements (weight, height, BMI (body mass index), waist circumference, waist to hip ratio (WHR)) every month.
Body mass index (BMI) was calculated as weight (in kilograms) divided by height (in meters squared). Based on the World Health Organization classification, overweight was defined as BMI between 25 and 29.9 kg/m2, and obesity was defined as BMI over 30 kg/m2. We also measured waist circumference (in centimeters) at the mid-point of the distance between the 12th rib and iliac crest and hip circumference at the greater trochanters with the legs brought together.
Arterial blood pressure was measured three times at the end of the physical examination with the subject in the sitting position. Participants whose average blood pressure levels were greater or equal to 140/90 mmHg or receiving antihypertensive medication were classified as hypertensive subjects .
Fasting blood samples were drawn between 7:00 a.m. and 10:00 a.m.
The biochemical analyses, including fasting serum lipids (total cholesterol (TC), triglyceride (TG), high-density lipoprotein-cholesterol (HDL-C)), glucose profile (fasting plasma glucose (FPG), fasting plasma insulin (FPI), glycated hemoglobin (HbA1c)), liver function tests (aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transpeptidase (GGT), alkaline phosphatase, bilirubin, albumin, total protein, International Normalized Ratio (INR)), were performed at baseline, 6 and 12 months with commercially available kits from Roche-Hitachi Systems which were analyzed on a Hitachi 917 autoanalyser. Low-density lipoprotein cholesterol (LDL-C) was calculated using the Friedwald formula (LDL-C = TC − TG/5 + HDL-C) .
Serum C-peptide was measured through an electrochemiluminescence immunoassay (Modular Analytics, Roche Diagnostics) with intra- and interassay coefficients of variation of 4.5% and 6.9%, respectively.
Insulin concentration was determined through RIA (Abbott Axsym System, Chicago IL), with intra- and interassay coefficients of variation of 4.5% and 6.9%, respectively.
FPI and C peptide were measured at baseline and 12 months. Insulin resistance (IR) was determined using Homeostasis model assessment (HOMA-IR)(fasting insulin level (mUI/l)x fasting glucose level (mg/dl)/405 ; a HOMA-IR index value of more than 2.0 was considered as the criteria of insulin resistance .
The oral glucose tolerance test (OGTT) was performed in patients with HbA1c higher than 5.5%. For OGTT, a glucose load equivalent to 75 g anhydrous glucose was given in a total water volume of 250 –300 ml . The glucose drink was consumed over 5 min. Timing for the rest of the test started at the beginning of ingestion. A further blood sample was collected 2 h after the glucose load in order to measure the glucose concentration. Diabetes diagnostic was made according to ADA 2003 criteria .
The definition of the metabolic syndrome (MetS) was based on the IDF criteria (central obesity defined as waist circumference over 94 cm in men, over 80 cm in women or BMI over 30 kg/m2 plus any two of the following factors: 1. triglycerides (TG) ≥1.695 mmol/l (150 mg/dl) or treatment; 2. high density lipoprotein-cholesterol (HDL-C) lower than 40 mg/dl in men, 50 mg/dl in women or treatment; 3. blood pressure ≥130/85 mmHg or medication; 4. fasting blood glucose ≥ 5.6 mmol/l (100 mg/dl) or medication for diabetes .
Liver fibrosis was non-invasively assessed using the Forns fibrosis index (FI) ; a value < 4.2 excludes liver fibrosis and a value > 6.9 is a predictor for significant fibrosis. Forns fibrosis index was calculated according to formula: 7.811–3.131 ln[platelet count (109/l)] + 0.781 ln[gamma-glutamyl transpeptidase (GGT) (UI/l)] + 3.467 ln[age (years)] − 0.014[cholesterol (mg/dl)]. The presence of significant fibrosis was predicted with a 96% negative predictive value (NPV) and 66% positive predictive value (PPV) .
The AST to platelet ratio index (APRI) was calculated by dividing the AST level (UI/l), expressed as the number of times above the upper limit of normal (ULN), by the platelet count (109/l): AST (/ULN) × 100/platelet count (109/l) . APRI is simpler to use than most of the other indices with similar performance to that of the Fibrotest (FT) and the Forns fibrosis index. This index has been validated in HCV patients . An 86% NPV and an 88% PPV were reported to predict the presence of significant fibrosis and a 98% NPV and a 57% PPV were reported to predict the presence of cirrhosis .
Hepatic steatosis (HS) was evaluated using ultrasonographic criteria. Hepatic ultrasound is a sensitive procedure for detecting liver fat (sensitivity 91–100, specificity 93–100) . The severity of echogenicity was graded as follows: grade 0, normal echogenicity; grade 1, slight, diffuse increase in fine echoes in liver parenchyma with normal visualization of diaphragm and intrahepatic vessel borders; grade 2, moderate, diffuse increase in fine echoes with slightly impaired visualization of intrahepatic vessels and diaphragm; grade 3, marked increase in fine echoes with poor or nonvisualization of the intrahepatic vessel borders, diaphragm, and posterior right lobe of the liver.
Estimated glomerular filtration rate (eGFR) was made according to CKD-EPI equation . The CKD-EPI equation, expressed as a single equation, is: GFR = 141 X min(Scr/κ,1)α X max(Scr/κ,1)-1.209 X 0.993Age X 1.018 [if female] X 1.159 [if black].
Independently done computer randomization was used to allocate numbers and divide the patients into two groups. Randomization was done by block design to ensure equal numbers in each group for every 4 subjects recruited.
All patients received nutrition counseling (NGLCD or LFD) in individual sessions every week in the first 6 months and every month thereafter until 12 months, with biological reevaluation at 6 and 12 months. All patients were required to submit a food journal at the baseline visit (before group allocation), as well as subsequent journals prior to the 6 month, 12 month, and each monthly visit. The food journal covers a 4 day period that includes 2 working days and 2 free days/weekend. Foods were measured using standard measuring cups and spoons and weight for 100 g; the input accuracy of the food journals was confirmed by using food-frequency questionnaires.
No supplements were allowed in this period. Patients who missed more than 30% of dietitian appointments or did not complete the food journals were considered noncompliant and were excluded from final analysis.
Macronutrient intake was calculated using the United States Department of Agriculture's food database (National Nutrient Database for Standard Reference, Release 16–1 and 17, Release dates July 2003 and 2004, Beltsville, MD) .
Subjects were required to limit alcohol intake to <20 g/week during the intervention period. Alcohol intake was averaged and recorded as grams per week.
Normoglucidic low-calorie diet
Dietitian doctors instructed participants to follow a diet comprising approximately 50-60% of daily caloric intake from carbohydrates , 25-35% of total calories from fat (less than 7% of total calories from saturated fat, less than 1% trans fatty acids, 10% monounsaturated fatty acids, 5-10% polyunsaturated fatty acids (PUFAs) and less than 300 mg cholesterol per day), proteins 15% of total calories (1.0 to 1.2 g/kg/day) , and <5% of caloric intake from simple sugars. Nutrient-rich choices that included whole grains, vegetables and fruit were prioritized. NGLCD was defined as a normoglucidic, normolipidic, normoproteic, low-calorie diet (100–500 kcal less than estimated energy needs).
Restriction of fat intake to 20% of total daily energy uptake with avoidance of trans-fat and saturated fat, up to 20% of the total calories from proteins and 60-65% carbohydrates. Further recommendation included increasing fibre uptake to 30 g per day, and avoiding liquid mono- and disaccharides. Moreover, patients were advised to consume at least 250 to 300 g of fruits, 125 to 150 g of vegetables, and 25 to 50 g of walnuts per day; in addition, they were also encouraged to consume 400 g of whole grains (rice, maize, and wheat) daily and to increase their consumption of olive oil. Compared with normoglucidic low-calorie diet, low-fat diet was defined by a low intake of fat (up to 20% of caloric intake), increased carbohydrate intake up to 60-65% of daily caloric intake, increased fiber intake (30 g/day), and protein intake up to 20%.
A healthy lifestyle includes regular physical activity (PA). Regular physical activity included 30 minutes of moderate intensity physical activities (e.g. brisk walking, jogging, cycling) for 3–7 days a week, recommended for persons with hepatitis C virus infection without advanced cirrhosis or other metabolic complications .
A high-energy diet is normally recommended for HCV-infected persons [19, 20]. Measured energy needs of patients with HCV infection, even in the absence of cirrhosis, are on average higher per unit of lean body mass than the needs of healthy individuals . The following provides two reasonable estimates of energy needs for patients undergoing physiological stress, such as those with infection: 25 to 40 kcal/kg, based on dry weight or an adjusted ideal weight  or add 20% to 40% to basal energy expenditure (BEE) using the Harris-Benedict equation .
In patients with overweight or obesity, the energy intake was individualized to be 100–500 kcal less than estimated energy needs because we designed it to induce at least a 5-10% weight loss at 6 months and to maintain this weight loss in the subsequent 6 months.
Results for continuous normally distributed data were expressed as mean ± standard deviations (SD). Tests of normality used were Kolmogorov-Smirnov with a Lilliefors significance correction and Shapiro-Wilk statistic. The comparison of mean value at baseline, 6 months and 12 months was performed with paired t-test. For continuous nonnormal distribution we used Wilcoxon's rank-sum tests, and data were reported as median ± interquartile ranges. Pearson's χ2 tests were used to compare changes in continuous variables from beginning categorical baseline characteristics. Wilcoxon's rank-sum tests were also used to compare changes in continuous variables from baseline to the 12 month. P-value less than 0.05 was considered significant. All statistical analyses were performed using SPSS 19 (copyright IBM).
The primary analysis was intention-to-treat and involved all patients who were randomly assigned . Two patients in the NGLCD group and eight patients in LFD groups were lost to follow up; thus data from 110 patients were available for the intention-to-treat analysis.