Effect of Short-term Annatto Carotenoids Supplementation on Oxidative Stress Status in Healthy Individuals

Study Overview
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Brief Summary
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Low-density lipoprotein (LDL) oxidation has a pivotal role in atherosclerosis development. There is a relationship between carotenoids serum concentration and cardiovascular (CV) benefits, mainly in oxidized LDL (oxLDL) reduction. Despite cardio protective effects of annatto carotenoids, bixin and norbixin, in vitro and in animal studies, its short or long-term supplementation effect on humans are not know. Objective: To analyse CV benefits of annatto carotenoids short-term supplementation in healthy individuals, comparing to lycopene effect. Methods: 16 healthy volunteers (8 men and 8 women) consumed 0.05 mg/kg b.w. of each treatment (bixin, norbixin, lycopene or placebo) through capsules, during 7 days. It was analysed the susceptibility of LDL to Cu2+-induced oxidation, biochemical parameters and oxidative stress biomarkers at the beginning and end of each treatment.

Detailed Description
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Graduate and post-graduate students were recruited in Federal University of Santa Maria to study participation. First, the health status of volunteers was analysed by questioner application (to evaluate lifestyle, family history, individual characteristics), anthropometric (height, weight, waist circumference) and biochemical parameters (glucose, lipid profile, transaminases, urea, creatinine) measurements. According to inclusion criteria, 18 volunteers were able to study participation, but just 16 people remained until the end of the study. Each treatment was composed by 7 capsules (containing 0.05 mg carotenoid or placebo/kg b.w.) which should be consumed once a day (preferably in the morning), during 7 days. It was conducted a randomized, double blind, placebo-controlled crossover clinical trial, where 16 participants received 4 proposed treatments (bixin, norbixin, lycopene and placebo) in different periods. To plasma, serum and red blood cells (RBC) obtainment, 2 fasting blood collections were made, in the beginning (Day 0) and the end (Day 7) of each treatment. These samples were used for evaluating the ex vivo oxidation of LDL induced by copper sulphate and biochemical and oxidative biomarkers measurements.

Official Title
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Short-term Annatto Carotenoids Supplementation Effect on LDL Susceptibility to Ex-vivo Oxidation and Oxidative Stress Biomarkers in Healthy Individuals

Conditions
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Oxidative Stress, Atherosclerosis

Intervention / Treatment
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* Dietary Supplement: Bixin
* Dietary Supplement: Norbixin
* Dietary Supplement: Lycopene
* Dietary Supplement: Placebo


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Normal levels of glucose, lipid profile, transaminases, urea and creatine Normal blood pressure, weight and body mass index (BMI) Exclusion Criteria: Chronic diseases (diabetes, dyslipiademia, hypertension cancer, etc.) Drug, alcohol and cigarette consumption/addiction Medication, vitamins or suplements consumption (except oral contraceptive used by women) Recent inflammatory/infectious diseases

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 35 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Prevention
Allocation: Randomized
Intervention Model: Crossover Assignment
Masking: Double


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: Bixin<br>Consumption of 0.05 mg bixin/kg b.w. through capsules (once a day) by 7 days in the morning. | Dietary Supplement: Bixin<br>* Volunteers consumed bixin capsules during 1 week , maintaining their usal lifestyle (avoiding excessive/unusual of fat and carotenoid-containing food, alcohol)<br>|
| Experimental: Norbixin<br>Consumption of 0.05 mg norbixin/kg b.w. through capsules (once a day) by 7 days in the morning. | Dietary Supplement: Norbixin<br>* Volunteers consumed norbixin capsules during 1 week , maintaining their usal lifestyle (avoiding excessive/unusual of fat and carotenoid-containing food, alcohol)<br>|
| Active Comparator: Lycopene<br>Consumption of 0.05 mg lycopene/kg b.w. through capsules (once a day) by 7 days in the morning. | Dietary Supplement: Lycopene<br>* Volunteers consumed lycopene capsules during 1 week , maintaining their usal lifestyle (avoiding excessive/unusual of fat and carotenoid-containing food, alcohol)<br>|
| Placebo Comparator: Placebo<br>Consumption of 0.05 mg placebo/kg b.w. through capsules (once a day) by 7 days in the morning. | Dietary Supplement: Placebo<br>* Volunteers consumed placebo capsules during 1 week , maintaining their usal lifestyle (avoiding excessive/unusual of fat and carotenoid-containing food, alcohol)<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Evaluation of LDL lipid moiety susceptibility to copper induced oxidation - oxidation rate | Change (Day 7 - Day 0) of oxidation rate of conjugated dienes formed after copper sulphate addition to LDL particles | Day 0 and Day 7 | 
| Evaluation of LDL protein moiety susceptibility to copper induced oxidation | Evaluate the change (Day 7 - Day 0) of loss of tryptophan fluorescence after oxidation induced by copper | Day 0 and Day 7 | 
| Evaluation of LDL lipid moiety susceptibility to copper induced oxidation - lag phase | Change (Day 7 - Day 0) of lag phase time of conjugated dienes formed after copper sulphate addition to LDL particles | Day 0 and Day 7 | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Evaluation of protein oxidation | Change at advanced oxidation protein products levels in plasma | Day 0 and Day 7 | 
| Evaluation of lipid oxidation | Change at malondialdehyde levels in plasma and red blood cells | Day 0 and Day 7 | 
| Evaluation of nitric oxide metabolites | Change at nitric oxide metabolites levels in serum | Day 0 and Day 7 | 
| Evaluation of plasma antioxidant capacity | Change at oxygen radical absorbance capacity in plasma | Day 0 and Day 7 | 
| Evaluation of enzymatic antioxidant defences | Change at antioxidant enzymes activities in red blood cells | Day 0 and Day 7 | 
| Evaluation of gluthatione cycle | Change at reduced glutathione/oxidized glutathione ratio in red blood cells | Day 0 and Day 7 | 
| Evaluation of red blood cell osmotic fragility | Change of red blood cell osmotic fragility after ex vivo exposure to membrane damage inducers | Day 7 | 
| Evaluation of carotenoids levels | Change at carotenoids levels in plasma and red blood cells | Day 0 and Day 7 | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Carotenoid, LDL oxidation, Cardiovascular disease, Antioxidant