Influence of the Fruit Matrix on Glucose and Insulin Responses After the Intake of Orange Juice in Healthy Human Males

Project proposal: Role of the fruit matrix on the absorption of glucose from orange juice in humans (ZULEMA) Background assumptions: Excessive consumption of free sugars can have negative health effects and, poor post-prandial control of glucose could be a marker for longer term type 2 diabetes risk. Sugars drive the glycemic index (GI) of beverages but, contrary to expectations, fruit juices and sugar-sweetened beverages provide different GI results despite often having the same total sugar content. This implies that natural and added sugars may have different effects on glycemic control, or that the combination of sugars within the juice matrix is somehow ameliorating the effect of the sugars on glycemic control. Research question: How does varying the matrix and source of sugars but not total sugars in fruit juices and drinks impact GI? The researchers assume that the free sugars will be the driver of GI but this may not be the case. Hypothesis: For the same amount of free sugars (sucrose, glucose and fructose) in juices and beverages, the fruits juice matrix (i.e. fiber, complex carbohydrates, citric acid, polyphenols and other phytochemicals, minerals, vitamins), will have a modulatory effect on the absorption of glucose. Objective: The primary objective of this study is to assess the effect of the presence of the juice matrix in 100% orange juice against manipulated juice drinks (to reduce the percentage of fruit matrix) on postprandial glycemia responses in healthy subjects. The secondary objective will be to characterize the effect on the insulin response. Experimental design: The glucose-responses will also be compared to that of glucose alone (Glycemic Index, GI). Methods: Juice preparation and logistics. The orange drinks will be produced and supplied by AMC Natural Drinks (Murcia, Spain). A 100% fruit juice (orange juice) will be compared with drinks containing 50%, and 0% of juice although with the same total sugar qualitative and quantitative composition (sucrose, fructose, glucose) as in the 100% juice. Chemical analyses: The researchers will characterize the sugar composition and the main fruit matrix components: Soluble and insoluble fiber chemical analysis. Sugars analysis. The juices will be manufactured and the sugar composition (sucrose, glucose and fructose) measured and adjusted to make sure that the sugar content does not changed during the storage period. Analyses will be performed by High Performance Liquid Chromtagraphy (HPLC) using the appropriate Refractive Index (RI) detectors. Phytochemicals analysis. Individual phenolics and other phytochemicals characteristic of orange juice. Flavanones, polymethoxyflavones, hydroxycinnamic acid derivatives, flavones, carotenoids, will be analysed using reversed phase HPLC with Ultraviolet-Diode Array (UV-DAD) detectors and Mass Spec (MS) detectors. Clinical trial: This study is a crossover, randomized, double-blind, dose-response intervention trial. All participants will repeat the study in separate times (to attain duplicate results). The study protocol has been already approved by the Catholic University Ethical Committee. Participants, Inclusion and Exclusion Criteria: The researchers will recruit male healthy adults aged 18 to 45 years of age via online classified advertisements, social media, and by word-of-mouth. Females are not eligible since menstrual cycle can influence glycaemic control. To meet the inclusion criteria, the participants will be required to have Body a Mass Index (BMI) between 18.5 and 29.9 kg/m2, to be habitual breakfast consumers, and to be willing to consume the study beverages. Participants will be excluded if they are on a diet or have food allergies or aversion to the drinks of the study. Furthermore, participants will be excluded if they had significant weight fluctuations within the past 6 months or a previous diagnosis of diabetes, pre-diabetes, gastrointestinal disease, liver disease, kidney disease, or a metabolic disorder. Sample size: The researchers estimated the sample size for a randomized crossover study in which the effects of the intervention (different test orange drinks) will be tested in a healthy adult sample population. For this purpose, the researchers used the free available software G\*Power 3.1.9.7 assuming a two-tails statistical power of 80% (1-β=0.80), a level of statistical significance of 5% (α=0.05) and an effect size of d=0.6. According to this, the minimum number of participants in the study should be N=24. These participants should also take part in the second round of tests (duplicate results). The final number of participants was increased up to 30 to count for potential withdrawals. Study Consent: A telephone screening interview will provide initial eligibility criteria. Participants who pass the telephone screening will be scheduled to attend an in-person information and final screening session at the laboratory to review study protocols and obtain written informed consent. The researchers will take some anthropometric measures and will explain to the participants the intervention and procedures during the whole study. The participants will also complete a series of general validated questionnaires about their dietary and lifestyle habits. Intervention Design: Eligible participants will complete four test sessions (+ two repetitions) at least 2-3 day apart. On separate mornings, participants will arrive at the laboratory at 8:00 am following a 10 to 12 h fast, except for a small amount of water which will be permitted up to 1 h before arriving. On the day before to each test session, the participants will complete a food diary to verify adherence to the polyphenol-free and citric fruits-free diet, fasting protocol, and to estimate the amount of nutrients consumed (principally, total carbohydrates, free sugars, fiber, and water). The participants will rapidly (within five minutes) consume one of the three drinks: (1) 100% orange juice, (2) orange beverage with 50% orange juice and 50% water with added sugars to match the content of the 100% juice, and (3) 100% water with added sugars to match the content of the 100% juice. Standard serving sizes (around 250 mL) will be calculated on the basis of total available carbohydrate to reach an intake of 25 g of free sugars. In addition, the participants will also consume a glucose solution (25 g) to estimate the GI. All test drinks will be served cold plastic containers with lids. Glycemic response will be measured at baseline (0 minutes), and 15, 30, 45, 60, 90 and 120 minutes post-consumption of the drink. After completion of the study, the participants will have a 2-3 weeks rest and will come back to the laboratory for a second test session. The aim of this repetition is to have amore robust analysis of the results and evaluate intra- and interindividual variability. Post-Prandial Glycemic Response: The researchers will collect blood samples by vein cannulation and will measure glucose by means of a Glucose Oxidase (GOD) Activity Assay Kit (E-BC-K520-M) (Elabscience, Texas, USA). Insulin measurement: Additional samples of blood will be collected from the vein. After centrifugation (1200-1500 ×g, 10 min, 4 ºC), the serum samples will be rapidly frozen and kept at -80 ºC until analysis of insulin levels. Insulin will be measured using a highly sensitive Human Insulin ELISA Kit (E-EL-H2665) (Elabscience, Texas, USA). Statistical Analyses: Statistical analyses will be performed using SPSS and R applying a Linear Mixed Model and post-hoc constrast to determine the effects and differences between the four interventions (i.e., glucose, 100% OJ, 50% OJ and 0% OJ) on blood glucose and insulin iAUC, Cmax, and T15min. A p-value \< 0.05 will be considered statistically significant.