We carried out a six-month, prospective, randomized placebo-controlled trial on a total of 155 postmenopausal women fulfilling the criteria of metabolic syndrome and at risk of breast cancer (with a familial history of breast or ovarian cancer, or women operated on for borderline lesions such as ductal hyperplasia or papillomatosis). All women were asked to follow a low-calorie diet and were randomly assigned to daily consumption of a combination of inositol and alpha lipoic acid or a placebo for six months.
The distribution of genetic and predisposing risk factors of breast cancer was 60% (93 pts) of patients with a familial history of breast cancer. Some 77% (71 pts) of them had a positive family history of breast cancer in a first-degree relative (mother, daughter, or sister), whereas 23% (22 pts) had a second-degree relative with a familial history of breast cancer. Some 64% (100 pts) of patients had a personal history of previous biopsies for borderline lesions. A total of 95% of women who underwent lumpectomy for borderline lesions had a familial history of breast cancer. Women were invited to attend a ‘high familial risk breast screening program’ set up by the National Cancer Institute. Women aged over forty had a clinical examination, mammogram and ultrasound. Women under forty had a clinical examination, ultrasound and MRI when necessary. Women were examined by the same doctors at all visits.
Measures and assay methods
In accordance with the Helsinki Declaration of 1975, after obtaining informed consent from each woman, anthropometric features were measured, including weight in kilograms, height in meters, waist and hip circumference, arterial blood pressure was taken and venous blood was collected on study entry. Body mass index (BMI) (kg/m2) was calculated from weight and height values and evaluated according to the World Health Organization classification (<25 kg/m2 = underweight/normal, ≥25 kg/m2 = overweight/obese). The waist-to-hip ratio (WHR) was obtained from waist and hip circumference, measuring the smallest circumference of both to discriminate between android and gynoid fat distribution. Fasting plasma glucose, insulin, HDL-C and triglycerides serum levels were assessed from blood samples. In particular, fasting plasma glucose, HDL-C and triglycerides were measured according to the NCEP ATP III criteria. Blood samples were locally assessed at the central laboratory of the National Cancer Institute. Sample collection was standardized by time at the blood withdrawing. Samples were taken in the early morning hours (between 8.00 and 10.00 am). Fasting plasma glucose assessment was measured by the COBAS INTEGRA Glucose HK cassette (GLUC2; Roche, Basel, Switzerland). It contains an in vitro diagnostic reagent system intended for use on COBAS INTEGRA systems for the quantitative determination of the glucose concentration in hemolysate. Electrochemiluminescence immunoassay (ECLIA) applied on Cobas 6000 was used for insulin concentration measurement. The enzymatic colorimetric test CHOD-POD was employed for cholesterol dosage. The GPO-POD method, based on the enzymatic determination of glycerol, using the enzyme glycerol phosphate oxidase (GPO), was used for triglycerides determination. Fresh, clear, unhemolyzed serum was the specimen of choice. The specimen was collected following the guidelines of NCCLS document H4-A3. Insulin levels were defined in the normal range when between 5 and 25 mcU/ml, whereas concentrations above 25 mcU/ml were considered corresponding to hyperinsulinemia. Insulin resistance was calculated by the homeostasis model assessment ratio-insulin resistance (HOMA-IR). To define women as positive for MS we adopted the Adult Treatment Panel III of the National Cholesterol Education Program criteria. All women presenting at least three of the five criteria described on ATPIII were considered affected by metabolic syndrome.
Baseline demographics of patients
A personal health questionnaire was assessed in order to measure personal health-related quality of life. Women were asked to give information about their daily alcohol intake, smoking, physical activities and sports. A total of 64% of women denied consuming any sort of alcoholic drink. Likewise 64% of women did not smoke cigarettes. On the other hand, only 17% of women answered that they did any physical activity and only 14% declared doing some sports.
Patients were invited to take part in the trial by their consultants. Women who agreed to take part in the trial were asked to sign a form. However, signing the form did not mean that they must remain in the trial.
After clinical examination was confirmed to be negative, a mammogram and ultrasound were performed. Women presenting a positive or suspicious mammogram and/or ultrasound were excluded from the trial.
Anthropometric measures, arterial blood pressure and venous blood samples were taken
Patients fulfilling at least three of the five criteria described on NCEP ATPIII (waist circumference (WC) >88 cm, HDL-C <50 mg/dl, blood pressure ≥130/85 mmHg, fasting plasma glucose ≥110 mg/dl, or triglyceride >150 mg/dl) were considered affected by metabolic syndrome. Patients affected by metabolic syndrome were considered eligible for enrollment.
All women were asked to follow a low-calorie diet and were randomly assigned to daily consumption of a combination of inositol and alpha lipoic acid (77 pts) or a placebo (78 pts) for six months. Patients enrolled were asked to follow the Therapeutic Lifestyle Changes (TLC) Diet according to the National Cholesterol Educational Program (NCEP) guidelines. The TLC eating plan is one that advises less than 7% of calories from saturated fat and less than 200 mg of dietary cholesterol per day. There should be no more than 25 to 35% or less of total daily calories coming from total fat intake. A limit of 2400 mg of day of sodium is recommended. The TLC diet recommends weight maintenance and avoidance of weight gain through caloric homeostasis. Reduction of saturated fat, trans fat, and cholesterol within the diet is recommended. Increased consumption of soluble fiber is requested.
Sample size (ss) was determined using the following formula [13
Z = Z value (for example 1.96 for 95% confidence level)
p = percentage picking a choice, expressed as a decimal (.5 used for sample size needed)
c = confidence interval, expressed as a decimal
Student’s paired t test was used for parametric variables and the Wilcoxon test for nonparametric variables like HOMA in order to analyze values at baseline and at the end point. One-way analysis of variance for parametric variables and the Kruskal-Wallis test for nonparametric variables like HOMA were used in order to compare treatment effects of the two arms. P <0.0005 was considered statistically significant.
The exclusion criteria were as follows: women presenting a positive or suspicious mammogram and/or ultrasound; women not fulfilling at least three of the five criteria of metabolic syndrome; diabetic women taking oral hypoglycemic drugs or insulin injections, diabetes diagnosed after a blood sample was taken in our institution, and patients consuming statins for hypercholesterolemia. As diabetes was considered an exclusion criterion, diabetes was diagnosed on laboratory determinations with fasting plasma glucose assessment ≥126 mg/dl according to American Diabetes Association guidelines . Fasting plasma glucose levels in the range between 110 and 126 mg/dl were considered as hyperglycemia.
The INOSIDEX trial was approved by the National Cancer Institute Ethical Committee, protocol number 23/12 OSS from Register M1/2 - metabolic syndrome, insulinemia, BMI in breast cancer prevention - a monocentric non-profit study from the National Cancer Institute of Naples - principal investigator: Dr. Immacolata Capasso. The primary investigators ensured that the study was conducted with full patient compliance, with protocol regulation conformity as well as international standards on human subject research. The primary investigators ensured compliance with institutional regulations as well as local and national law.
This phase comprised of six-monthly visits. Women were examined by the same doctors at all visits. During each visit, a clinical examination was performed, anthropometric features and arterial pressure were measured, blood samples were taken, dietary questionnaires were compiled to understand patient compliance to the study. Health status and medication changes were recorded and adverse events were asked for.