This study is a 6-month, prospective, randomized, open-label trial, with parallel arms and blinded endpoints. Subjects were allocated in a 1:1 ratio to each arm of the study treatment. The trial was registered on 26/07/2017 at the clinicaltrials.gov (NCT03230123), under the acronym of BIOMEL Study (Effects of Green Banana BIOmass Consumption in Patients with Pre-diabetes and Diabetes MELlitus) and can be accessed through the internet (https://clinicaltrials.gov/)9. The study protocol is in agreement with the Ethical Principles for Medical Research Involving Human Subjects as stated by the Declaration of Helsinki. This trial was approved by local Human Ethics Committee and all patients signed the written informed consent before data collection. For more details about the design see Costa et al.9.
The study was conducted at the Department of Medicine, Federal University of Sao Paulo, SP, Brazil. The recruitment process began in February 2016, and the intervention was conducted until March 2017. Briefly, patients of both sexes, aging 60–72 years, with diabetes (glycated haemoglobin ≥ 6.5%) and pre-diabetes (HbA1c between 5.7% and 6.4%, or with confirmed type 2 diabetes), receiving a stable dose of anti-hyperglycemic drugs were eligible9. For this subanalysis, we kept the same inclusion/exclusion criteria. We did not use the fasting glucose cut off of 126 mg/dL for inclusion because the patients were under a stable dose of anti-hyperglycemic drugs.
They were assigned to either diet intervention plus green banana biomass or diet intervention alone in a 1:1 ratio, using a random-number generator program. Thirteen patients were discontinued due to their need for insulin (six in banana group, seven in control), and five subjects in control group did not comply with the diet intervention. The final number were 21 in banana group and 18 in control group.
Patients under insulin therapy, or those that during the study needed change in dose or addition of medication for diabetes were excluded. Neoplasms, except basal-cell carcinoma, heart (NYHA class III or IV) and renal failure (e-GFR < 30 mL/min) or dialysis therapy, AIDS, uncontrolled hypothyroidism (TSH > 10 μUI/mL), active liver disease, severe psychiatric disorders, or any other disease that, in the investigator’s opinion, could interfere with the results were also excluded. Investigators who performed all analyses were blinded to interventions.
The calculation of sample size took into account variations in glycated haemoglobin (HbA1c), with a type I error alpha of 0.05 and a type II error beta of 0.2 (80% power)9. For this subanalysis, a convenience sample was used.
Dietetic parameters
The diet plan was individualized according to the Brazilian Society of Diabetes, taking into account the total energy expenditure (TEE), with standardized menus for weight reduction (20–25 kcal/kg of current weight), weight maintenance (25–30 kcal/kg of current weight), and weight gain (30–35 kcal/kg of current weight), according to patient characteristics17. Macronutrients intake was accessed by the Acceptable Macronutrient Distribution Range18. Substitutions by equivalent foods were made using a food replacement list (FRL)19.
Green-banana biomass
Green-banana biomass was added to any food preparation without heating as 2 tablespoons (40 g) per day, providing ~ 4.5 g of RS. According to the manufacturer, nutrition information for green banana biomass (per 20-g portion or one tablespoon) is: carbohydrates 2.83 g, proteins 0.18 g, fiber 1.12 g, total energy expenditure 12 kcal. The resistant starch content is ~ 12%. It does not contain significant amounts of sodium and total fat, saturated, or trans fatty acids9,20. The product was well tolerated and did not cause discontinuation due to side effects.
Evaluation of food consumption
Food consumption was estimated by 24 h food records and standardized food-frequency questionnaires (FFQs) obtained at baseline (T0) and 6-months (T6), with total energy intake, macro- and micro-nutrients, lipids, cholesterol, carbohydrates, fatty acids, and vitamins calculated by the Avanutri Software (Avanutri Revolution, v. 4.0)21,22,23.
Biochemical analysis
Fasting blood samples were obtained from all participants at T0 and T6. Commercial kits (Cobas Mira, Roche, Switzerland) were used in the analysis for blood glucose, total cholesterol (TC), glycated hemoglobin (HbA1c, %), HDL-C, and triglycerides (TG). LDL-C concentration was calculated by the Friedewald equation24. Ox-LDL concentration (mU/L) was determined using Mercodia Oxidized LDL—ELISA kit (Mercodia AB, Uppsala, Sweden) according to the manufacturer’s instructions. Fasting insulin was measured using the immunofluorometric assay. The HOMA-IR [fasting insulin (μUI/mL) × fasting glucose (mg/dL)/405] was calculated, with the cutoff value set at ≥ 2.825.
Protein concentrations in LDL particles were determined by using the bicinchoninic acid (BCA) method, Pierce BCA Protein Assay kit (Thermo Fisher Scientific, MA, USA) with bovine serum albumin (BSA) as standard26.
Low-density lipoprotein separation
Blood was collected and immediately separated in plasma and stored at − 80 °C until analysis. Low-density lipoprotein was isolated from plasma by preparative sequential ultracentrifugation (18 h, 4 °C, 105.000×g), using a density cut-off point of 1.063 g/mL, by ultracentrifuge equipped with a fixed-angle rotor (Hitachi Himac CP 70MX, Tokyo, Japan)27. These samples were dialyzed against PBS with EDTA (pH 7.4, 4 °C, 12 h, with agitation) to remove the salts.
Z-scan technique
The Z-scan (ZS) is an experimental technique to measure nonlinear optical properties of materials28. In this technique, the LDL solution sample was encapsulated between two micro-slides glass with a spacer of 200 μm and is illuminated by a focused laser beam (wavelength 532 nm, power 100 mw), propagating in the z direction. A mechanical chopper provides light pulses of about 30 ms. The sample moves along the z-axis before and after the focal point z = 0 and the transmitted-laser beam is detected by a detector. The normalized transmittance as a function of the sample-z position is calculated dividing the voltage on the photodetector at each z-position of sample by the voltage when the sample is at a position far from the focal point. More details about the setup and data treatment can be found in our previous works11,29. The typical result in the ZS experiment is a valley to peak (or peak to valley) curve. This peak to valley amplitude (\(\Delta \Gamma_pv\)) is proportional to the phase shift (θ) of the thermal lens formed. In this study, we normalized the values of θ to compare the results from various patients because each sample had different LDL concentrations.
UV–visible spectroscopy
The spectrophotometer measures the intensity of light passing through a sample and compares it to the intensity of the incident light beam. The linear-absorbance spectra were measured by a UV–visible spectrophotometer with light wavelength from 200 to 900 nm, using deuterium and tungsten halogen light sources and a spectrometer (USB4000, from Ocean Optics) connected to a computer for data acquisition. The samples were conditioned into a quartz cuvette, with optical path length of 1 cm. The extinction spectrum from the spectrophotometer is the sum of both the Rayleigh scattering and the absorbance. The Rayleigh scattering is proportional to λ−4, and its intensity is estimated for each one of the samples29. The absorbance is calculated by removing the scattering contribution from the extinction spectra. As is known, the LDL particle contains various molecules, and each of them has different absorption spectra16. For instance, the maximum light absorption of the ApoB-100 is at λApo ≈ 280 nm30,31, the cholesterol at λchol < 200 nm32, the α-Tocopherol at λα-Toc ≈ 210 nm and the carotenoids at λβ-Car ≈ λα-Car ≈440 and 480 nm33,34. In the present study we smeasured the absorbance values of LDL solution samples at the wavelength corresponding to the maximum of the absorbance spectrum of Carotenoids, λ = 480 nm, at baseline and after 6-months treatment.
Small angle X-ray scattering (SAXS)
Small-angle X-ray scattering (SAXS) is a standard technique that can be used to the study of particles in solution, providing information about size, polydispersity, shape, oligomerization, flexibility and aggregate state35,36. SAXS data were collected in a Xenocs-XEUSS diffractometer. X-rays (wavelength λ = 1.54 Å Cukα) are collimated by two sets of scatter-less slits and reaches the LDL sample placed in a cylindrical borosilicate glass capillary. This capillary is mounted on a homemade stainless-steel case, which allows an easy handling, wash and rinse. Therefore, the lipoproteins and the corresponding buffers can be measured in the same conditions. The two-dimensional scattering patterns were registered by a detector. The images were integrated with the Fit2D software and the data treatment was performed using standard procedures35. As a result, one obtains the scattering intensity as a function of the reciprocal space moment transfer modulus, q, defined as q = 4πsin(θ)/λ, where 2θ is the scattering angle. The data was collected at sample to detector distance of 0.90 m and the available q range is 0.015 < q < 0.45 Å−1.
Dynamic light scattering (DLS)
The DLS, known as photon correlation spectroscopy, was used to assess eventual aggregation of particles and the LDL size distributions37. DLS measurements were carried out using a 90Plus Particle Size Analyser (Brookhaven, Holtsville, NY, USA). In this technique, the sample is illuminated by a laser beam (wavelength 657 nm and power of 35 mW) and the fluctuations of the scattered light are detected by a fast photon detector positioned at 90° from the incident light direction. The DLS measurements provide intensity correlation functions that are analyzed to determine the particle-size distribution, weighted by volume, number, and intensity of scattered light38,39. The fits shown in this work were obtained by using the NNLS (non-negative least squares) method40.
Lipoprint system
The lipoprotein fractions (VLDL and IDL) and sub-fractions of LDL were determined by the Lipoprint system (Quantimetrix, Redondo Beach, CA), which is based on the separation and quantification of lipoprotein sub-fractions by non-denaturing polyacrylamide tube gel electrophoresis. To perform this procedure, 25 μL of the serum or plasma was added to the polyacrylamide gel tube and 200 μL of the dye-gel solution. The sample was homogenized. Then the tubes containing the samples were photo-polymerized and subjected to the electrophoresis process. After separation of the sub-fractions, the tubes were scanned in order to identify each subclass41. The LDL-1 and LDL-2 subclasses were classified as Large LDL and subclasses LDL-3 to LDL-7 were classified as smaller and denser particles (Small LDL). Results are shown in percentage (%) and concentration (%/total cholesterol level) of subclasses. The LDL phenotypes were based in cut-off points (phenotype A ≥ 26.8 Å and phenotype non-A < 26.8 Å)13,14,42. All analyses were conducted in duplicate and coefficients of variance intra and inter assay were 1–15%.
Statistical analysis
Numerical variables were expressed as means ± Standard Deviation (SD) for normal distribution and median [Inter-Quartile Range (IQR = Q1–Q3)] for non-normal distribution. Shapiro–Wilk test was used to verify normality of the data distribution. For comparison between groups, unpaired two sample T test, for normal distribution or Mann–Whitney test, for non-normal distribution, were used. Within-group comparisons were carried out using the paired sample T test, to compare groups with normal distribution, or Wilcoxon signed-rank test for non-normal distribution. Statistical significance was set at p value < 0.05.
Ethical approval
The study protocol was approved by the local Ethics Committee (Comitê de Ética em Pesquisa da Universidade Federal de São Paulo, CEP-UNIFESP, CAAE: 48,643,415.2.0000.5505).
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