Lutein is a xanthophyll pigment found in photosynthesizing organisms. Its beneficial effects on health, including brain function and visual performance, have been recognized for many years. However, to date, only a limited number of well-designed human intervention studies have been published regarding the effects of incorporating lutein into the daily diet (as humans are unable to synthesize lutein endogenously). Unfortunately, within the Western dietary pattern, the average daily intake of lutein is approximately 1.7 mg, whereas achieving health benefits- such as reducing the risk of age-related macular degeneration and cataract, as well as neurodegenerative diseases- requires an intake of 6 to 14 mg per day. In light of the above, it is therefore justified to enrich the daily diet with products that are sources of lutein. Unfortunately, the consumption of dark green vegetables- the richest dietary source of lutein-remains insufficient. Lutein preparations currently available on the market are extracted from marigold or calendula flowers. However, lutein production from these raw materials requires greater water consumption and land use, which is not aligned with the principles of sustainable development. An alternative approach involves the production of lutein preparations from microalgae (from species approved as food by the European Food Safety Authority, EFSA), as the rate of lutein production from microalgae is three to six times higher than that from marigold flowers. Taking the above into account, the primary objective of this project is to evaluate the dose-response relationship in establishing the anti-aging mechanism of action of lutein derived from microalgae. As part of the project, a six-month randomized, placebo-controlled trial is planned. The study will include 300 polish women (aged 48-60 years), after natural menopause, with visceral obesity (waist circumference ≥ 88 cm). In order to enable the inclusion of such a large study population, the research team will conduct intensified recruitment efforts for several months prior to the initiation of the dietary intervention. Participants who meet the inclusion criteria will be randomly assigned to one of three groups: lutein supplementation at a dose of 6 mg (n = 100), lutein supplementation at a dose of 14 mg (n = 100), or placebo (n = 100). The volunteers will be instructed to take the prescribed preparation daily for 180 days. All preparations will be identical in appearance, taste, and smell. All volunteers expressing willingness to participate in the experiment will be asked to provide written informed consent prior to enrollment in the study. The study will be conducted in accordance with the previously calculated sample size (n = 300 postmenopausal women with visceral obesity). In order to obtain a homogeneous study population, appropriate inclusion and exclusion criteria will be applied. Conducting the research in such a large and homogeneous group will enable the generation of high-quality scientific evidence identifying the dose of microalgae-derived lutein that allows for the determination of its anti-aging mechanism of action. It will also be possible to elucidate the potential role of short-chain fatty acids (SCFAs) in feces in this process, which may represent a significant novelty in this field of research. The study will contribute to the development of new knowledge regarding the anti-aging properties of lutein derived from microalgae in populations vulnerable to cognitive impairment (postmenopausal women). However, microalgae-derived lutein may be used as a dietary supplement not only in the studied group but also in the general population. The specific objectives are as follows: * To assess the effect of lutein derived from microalgae (at doses of 6 mg and 14 mg) on the concentration of brain-derived neurotrophic factor (BDNF), inflammatory markers, cardiometabolic parameters, fecal short-chain fatty acid (SCFA) concentrations, and cognitive function in postmenopausal women with visceral obesity. * To evaluate adherence to lutein supplementation recommendations (by assessing macular pigment optical density). * To determine whether supplementation with microalgal lutein increases fecal SCFA concentrations and whether SCFAs may act as mediators in improving inflammatory markers and cognitive function in postmenopausal women with visceral obesity. * To determine whether supplementation with microalgal lutein (at doses of 6 mg and 14 mg) affects changes in selected gut bacterial populations and β-glucuronidase enzymatic activity in postmenopausal women with visceral obesity. * To analyse the association between polymorphisms in genes related to lutein metabolism and transport in the body and the effectiveness of supplementation with this compound. * To identify dietary behaviour patterns associated with high exposure to bisphenol A (BPA) and the presence of inflammation in postmenopausal women with visceral obesity.
The primary endpoint will include the assessment of: * cognitive function; * macular pigment optical density; * inflammatory markers (IL-6, IL-10, IL-1β, TNF-α, β-amyloid); * blood estrogen concentration; * levels of brain-derived neurotrophic factor (BDNF); * the abundance of selected gut bacterial populations and β-glucuronidase activity; * polymorphisms in genes associated with lutein metabolism and transport. The secondary endpoint will include the assessment of: * cardiometabolic parameters (lipid profile: total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides; fasting glucose and insulin); * concentrations of gut microbiota metabolites in feces (short-chain fatty acids; SCFAs); * anthropometric parameters and bone mineral density; * arterial blood pressure. At the screening stage, a brief Montreal Cognitive Assessment (MoCA) will be administered to ensure the inclusion of participants who do not exhibit symptoms of dementia. Follow-up visits will be conducted at four-week intervals. Anthropometric parameters (body weight, waist circumference, and body composition) will be measured at each follow-up visit. Cognitive function will be assessed using selected standardized tests from the CNS Vital Signs battery before the initiation and after completion of the intervention. Biochemical blood parameters, including inflammatory markers (IL-6, TNF-α, IL-1β, IL-10), insulin, brain-derived neurotrophic factor (BDNF), β-amyloid, lipid profile, glucose, and estrogen concentrations, will be measured before and after the intervention. An additional 5 mL blood sample will be collected for the assessment of gene polymorphisms in CD36 (rs1761667) and BCO1 (rs6564851, rs12934922, rs7501331). Fecal short-chain fatty acids (SCFAs) and β-glucuronidase activity will also be assessed before and after the intervention. Bacterial DNA will be isolated from stool samples using the QIAamp DNA Mini Kit according to the manufacturer's instructions, and the abundance of selected gut bacteria will subsequently be determined. The remaining biological material will be stored at -20°C (biobank) pending further analyses. Macular pigment optical density (MPOD) will be assessed before and after the intervention using the computerized Macular Pigment Screener MPS II. In addition, prior to the intervention, participants will be required to complete a 24-hour urine collection for the determination of bisphenol A (BPA) concentration. Adherence to supplementation recommendations and tolerance of the lutein supplement will be evaluated at each follow-up visit using a questionnaire. The tested supplement will consist of capsules containing "Green Chlorella Prime" powder, manufactured by the French company Biorea. The company is authorized to produce dietary supplements in accordance with ISO 22000/FSSC and GMP+ standards. The FSSC 22000 certificate is an international food safety standard that protects both companies and consumer health. The supplement used in the study will contain lutein derived from Chlorella sorokiniana and will be produced using optimized technological processes to obtain a microalgal supplement with a high lutein content. The study will be conducted in a double-blind manner; neither the participants nor the research staff conducting the assessments will be aware of group allocation. All participants will be instructed to take the prescribed dose daily for 180 days. Compliance with supplementation will be verified by measuring macular pigment optical density. Participants will be asked to record supplement intake and return empty packaging at each follow-up visit. Supplement tolerance will be assessed during each visit using a questionnaire. Assessment of Cognitive Function Neuropsychological evaluation will be conducted before and after the intervention using the CNS Vital Signs clinical test battery, comprising seven tests assessing verbal memory, visual memory, psychomotor speed, cognitive flexibility, complex attention, processing speed, reaction time, executive functioning, and reasoning. Assessment of Macular Pigment Optical Density Macular pigment optical density will be assessed before and after the intervention using the computerized Macular Pigment Screener MPS II. Dietary Assessment Dietary intake will be evaluated before and after the intervention using the KomPAN questionnaire, supplemented by four 24-hour dietary recalls. Nutritional Status Assessment Nutritional status assessment will include baseline determination and monitoring of selected anthropometric parameters (body weight, body composition including visceral adipose tissue, waist and hip circumference, bone density) as well as selected biochemical blood parameters. Height will be measured using a medical stadiometer to the nearest 0.5 cm under standardized conditions. Body weight will be measured to the nearest 0.1 kg with participants standing barefoot in light clothing. Body composition will be assessed using dual-energy X-ray absorptiometry (DXA), a safe and non-invasive method based on the differential attenuation of ionizing radiation by tissues of varying density. Fat mass (including visceral fat), lean mass, and bone mineral density will be measured. Waist and hip circumferences will be measured using a non-elastic measuring tape according to standardized anatomical landmarks. All anthropometric measurements and assessments of nutritional and dietary status will be conducted at the Department of Human Nutrition and Dietetics, Faculty of Food Science and Nutrition, at the Nutrition Research Center. Biochemical Blood Parameters Among enrolled participants, the following biochemical blood parameters will be determined: fasting glucose and insulin; lipid profile (total cholesterol, LDL-C, HDL-C, triglycerides); IL-6, IL-10, IL-1β, TNF-α, β-amyloid; BDNF; and estrogen levels. Blood samples will be collected by a qualified nurse at the Nutrition Research Center of the Department of Human Nutrition and Dietetics, Poznań University of Life Sciences. Blood will be drawn twice (before and after the intervention) from the antecubital vein in a fasting state into clot-activator tubes (18 mL + 5 mL for genotyping). Serum will be obtained by centrifugation. Insulin, inflammatory markers, β-amyloid, BDNF, and estrogens will be measured using ELISA assays, whereas glucose and lipid profile parameters will be analyzed using a Konelab biochemical analyzer. Biological material for further analyses will be stored at -80°C. Genomic DNA Isolation Genomic DNA will be isolated from peripheral blood using commercial TaqMan assays. Genotyping will be performed using Real-Time PCR, combining PCR amplification with fluorescent probe detection to identify single nucleotide polymorphisms. An additional 5 mL of whole blood collected into EDTA tubes will be required for DNA isolation. Determination of Bisphenol A (BPA) in Urine Prior to the intervention, each participant will complete a 24-hour urine collection. BPA concentration will be determined using ultra-high-performance liquid chromatography with diode-array detection (UPLC-DAD). Determination of Short-Chain Fatty Acids in Feces Participants will provide stool samples at baseline and at the final visit. Quantitative determination of SCFAs will be performed using gas chromatography with flame ionization detection (GC-FID) at the Department of Bromatology, Poznań University of Medical Sciences. Assessment of β-Glucuronidase Activity A portion of the stool sample will be homogenized in buffer solution, and β-glucuronidase activity will be determined based on the hydrolysis of a specific substrate to a fluorescent product, both before and after the six-month intervention. Biobank Preparation and Microbiota Analysis Fresh bacterial DNA will be isolated from stool samples using the QIAamp DNA Mini Kit according to the manufacturer's protocol and stored at -20°C for future in-depth microbiota analyses. The abundance of selected gut bacteria (e.g., Bifidobacterium longum, Akkermansia muciniphila, Faecalibacterium prausnitzii) will be determined by PCR. Participants will be recruited through public advertisements inviting volunteers to participate in scientific research and will be enrolled after receiving detailed information regarding the study objectives, protocol, measurements performed, and study duration.
Study Type
INTERVENTIONAL
Allocation
RANDOMIZED
Purpose
PREVENTION
Masking
TRIPLE
Enrollment
300
Participants receive oral lutein supplementation administered daily for 180 days. Two dosing regimens are used in the study: 6 mg/day and 14 mg/day. The supplement is identical in appearance, taste, and smell to the placebo preparation.
Participants receive an oral placebo preparation administered daily for 180 days. The placebo is identical in appearance, taste, and smell to the lutein supplement.
Faculty of Food Science and Nutrition, Poznan University of Life Sciences
Poznan, Poland
Serum IL-6 concentration [pg/ml] by ELISA
Serum IL-6 will be analysed by the accredited laboratory by Enzyme-Linked Immunosorbent Assay (ELISA) quantitatively and will be expressed in pg/mL
Time frame: From enrollment to the end of treatment at 6 months
Serum concentration of Brain-Derived Neurotrophic Factor (BDNF) [pg/mL]
The BDNF concentration will be assessed from serum by ELISA method in the lab of Department of Human Nutrition and Dietetics, Poznan University of Life Sciences.
Time frame: From enrollment to the end of treatment at 6 months
Macular Pigment Optical Density (MPOD) measured in density units [d.u.]
MPOD will be measured by the non-invasive optical measurements (lutein accumulates in the macula) based on heterochromatic flicker photometry technique.
Time frame: baseline and after 6 months of the intervention
Neurocognitive Index score measured by CNS Vital Signs
To assess cognitive function, we will use the CNS Vital Signs neurocognitive test battery. The core 'BRIEF-CORE' battery consists of seven subtests: Verbal Memory (VBM), Visual Memory (VIM), Finger Tapping (FTT), Symbol Digit Coding (SDC), Stroop Test (ST), Continuous Attention Test (SAT), and Continuous Performance Test (CPT). These subtests provide quantitative scores across multiple cognitive domains, including complex memory, visual memory, verbal memory, psychomotor speed, motor speed, processing speed, reaction time, cognitive flexibility, executive function, complex attention, and simple attention. Cognitive function will be compared between baseline (pre-intervention) and post-intervention assessments to identify potential changes in performance.
Time frame: From enrollment to the end of treatment at 6 months
Estrogen concentration [pg/ml] by ELISA.
Estrogen concentrations \[pg/ml\] will be assesed from vascular blood sample at the Poznań University of Life Sciences by Enzyme-Linked Immunosorbent Assay (ELISA).
Time frame: From enrollment to the end of treatment at 6 months
Gene polymorphisms CD36 (rs1761667), BCO1 (rs6564851, rs12934922, rs7501331) in blood
The genomic DNA will be isolated using ready to use kits according to manufacturer protocols. The polymorphism of UGTs genes will be performed by real-time PCR using a TaqMan probe assay and a LightCycler 480II following the manufacturer's protocol.
Time frame: From enrollment to the end of treatment at 6 months
Serum Interleukin-1 beta (IL-1β) concentration [pg/ml] by ELISA
IL-1β will be analysed by accredited laboratory by ELISA method. The result will be quantitative and expressed in pg/mL.
Time frame: From enrollment to the end of treatment at 6 months
Tumor necrosis factor α (TNF-α) concentration [pg/ml] by ELISA
Serum TNF-α will be analysed in accredited laboratory by the ELISA method. The results will be quantitative and expressed in pg/mL
Time frame: From enrollment to the end of treatment at 6 moths.
The abundance of selected gut bacteria determined by PCR
Using the bacterial DNA obtained from stool samples, the abundance of selected gut bacteria (i.e. Bifidobacterium longum, Akkermansia muciniphila, Faecalibacterium prausnitzii) will be determined using PCR.
Time frame: From enrollment to the end of treatment at 6 months
SCFA concentration [µmol/g] by GC-FID
SCFA concentration \[µmol/g\] will be assessed using gas chromatography with flame ionisation detection (GC-FID). The research will be carried out at the Department of Bromatology, Poznań University of Medical Sciences.
Time frame: From enrollment to the end of treatment at 6 months
β-glucuronidase activity [U/g stool] assessed by β-glucuronidase Activity Assay Kit (Fluorometric)
A faecal sample will be dissolved in a buffer solution and homogenised, and β-glucuronidase activity will be determined based on the hydrolysis of a specific substrate into a fluorescent product. The β-glucuronidase assay will be performed both before and after the completion of the 6-month experiment.
Time frame: From enrollment to the end of treatment at 6 months
Fasting blood glucose concentration in mg/dl
Fasting blood glucose concentration measured in venous blood samples using standard laboratory methods.
Time frame: From enrollment to the end of treatment at 6 months
Total cholesterol (TC) concentration in mg/dl
Serum TC concentration measured using standard laboratory methods.
Time frame: From enrollment to the end of treatment at 6 months
Low-density lipoprotein (LDL) cholesterol concentration in mg/dL
Serum LDL concentration measured using standard laboratory methods.
Time frame: From enrollment to the end of treatment at 6 months
High-density lipoprotein (HDL) cholesterol concentration in mg/dL
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Serum HDL concentration measured using standard laboratory methods.
Time frame: From enrollment to the end of treatment at 6 months
Triglyceride (TG) concentration in mg/dL
Serum TG concentration measured using standard laboratory methods.
Time frame: From enrollment to the end of treatment at 6 months
Body weight [kg]
Body weight is measured in a standing position, without shoes, in light clothing, to an accuracy of 0.1 kg.
Time frame: From enrollment to the end of treatment at 6 months
Body height [cm]
Body height will be measured using a medical anthropometer to an accuracy of 0.5 cm. The measurement will be taken barefoot, in a standing position - without excessive tension in the body, shoulders relaxed, upper and lower limbs freely extended, feet together with heels touching and weight evenly distributed, head positioned in the eye-ear plane (Frankfurt plane; the upper edges of the external auditory meatus and the lower edge of the orbit at the same level).
Time frame: From enrollment to the end of treatment at 6 months
Waist to hip ratio [-] by non-elastic measure tape
Hip and waist circumferences will be taken using a non-stretch tape measure \[cm\]. Waist circumference measurement - the tape measure should be placed horizontally or at a slight angle halfway between the lower edge of the ribs and the upper edge of the iliac crest. The measurement should be taken whilst holding one's breath. Ratio between the waist and hip circumference will be taken.
Time frame: From enrollment to the end of treatment at 6 months
Body composition (visceral fat, lean muscle mass and bone density) [kg] measured by DXA
Body composition will be measured using dual-energy X-ray absorptiometry (DXA), a method that utilises the attenuation of ionising radiation as it passes through tissues of varying density. This method is safe and non-invasive. It measures body fat mass, including visceral fat, lean body mass and bone density.
Time frame: From enrollment to the end of treatment at 6 months
Bisphenol A (BPA) concentration [μg/L] by high-performance liquid chromatography (HPLC)-Diode Array Detector (DAD)
Detection by DAD will be carried out at a wavelength of approximately 227 nm, corresponding to the maximum absorbance of the phenolic chromophore of BPA. The injection volume will be 20 μL. Under these conditions, a symmetrical BPA peak with sufficient retention time and separation from interfering matrix components will be obtained. A calibration curve will be prepared based on BPA standards in the range of 0.1-50 μg/L, prepared in a solvent appropriate for the sample matrix. Peak areas will be analyzed relative to the standard concentrations, and linearity will be assessed using linear regression. The limit of detection (LOD) will be determined at a signal-to-noise ratio (S/N) of 3, and the limit of quantification (LOQ) at an S/N of 10. The method will be evaluated in terms of precision, accuracy, linearity, specificity, LOD/LOQ, and analyte recovery from the matrices.
Time frame: baseline only
Food frequency intake
Food frequency intake will be assessed by using a validated questionnaire and will include questions concerning food groups with six possible answers: (1) never, (2) 1-3 times a month, (3) once a week, (4) 2-3 times a week, (5) once a day, (6) a few times during the day. Those responses will be converted into an average frequency of food intake per day \[times per day\].
Time frame: From enrollment to the end of treatment at 6 months.
Nutrition intake
Nutrition intake will be assessed by three day dietary diary. The diary will be verified by the trained dietitian during the nutrition interview in lab of the Department of Human Nutrition and Dietetics, Poznan University of Life Sciences.
Time frame: From enrollment to the end of treatment at 6 months.