Differences in gut microbiota profile between women with active lifestyle and sedentary women methods
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Materials and methods
Forty premenopausal women were recruited according to inclusion criteria: age 18-40 years and body mass index (BMI) 20-25 kg/m 2. Criteria for physical exercise were as follows: sedentary women who did not perform the minimal exercise established as healthy by WHO, ie 3 days of exercise per week for 30 minutes at a moderate intensity [ ]; and for active women, those who performed at least 3 hours of physical exercise per week. Exclusion criteria were any kind of pathology, previous gastrointestinal surgery, antibiotics intake during three months prior to the study, smoking, prebiotics, probiotics, vegetarian or vegan, nutritional or ergogenic complements, pregnancy or lactation. All subjects were Caucasian. The Ethics Committee for Clinical Research of the Ramón y Cajal Hospital (Madrid, Spain) approved the study. Written and informed consent was obtained from all pa...
Physical activity and sedentary time determination
Levels of physical activity performed by both groups of women were measured with an ActiSleep V.3.4.2 accelerometer. (Actigraph, Manufacturing Technology Inc., Shalimar, FL). This allowed us to verify in an objective manner the presorting of sedentary and active individuals as it determines body movements in all three axes. Acceleration, energy expenditure, intensity of physical activity and body position was recorded. Results were analyzed with Actilife software (Actilife6, Actigraph). Recordings were performed for 7 days (5 weekdays and 2 weekend days). Participants were instructed to wear the accelerometer on the right wrist all day except when they had a shower or performed pool activities. Data were considered valid when they contained a record of at least five valid days including at least one weekend day, and at least 10 hours of activity were recorded. The time-sampling interv...
Stool collection, DNA extraction and sequencing
Participants were provided with a plastic device to collect stool samples, which were stored at -80°C until extraction. DNA was extracted using the commercial Power Soil DNA Kit (Mobio, CA) with a bead-beating homogenizer (Bullet Blender® Storm, Next Advance, NY). The concentration and purity of DNA was measured using the Quant-iT PicoGreen dsDNA Assay Kit (ThermoFisher Scientific, Waltham, MA).
Quantitative PCR analysis
Quantitative PCR (qPCR) analysis was carried out on a CFX Connect™ Real-Time PCR Detection System (BioRad, Barcelona, Spain) using SYBR Green I chemistry (BioRad) in 20 µL reactions containing one microliter (1-10 ng) of DNA template and 200 nM of primers. Cycling parameters were 95 °C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, 1 minute at the established annealing temperature and 72°C for 45 seconds. Subsequently, melting curve analysis was performed, in which fluorescence was measured as the temperature increased from 50°C to 95°C. For bacterial quantification, standard curves were made using serial dilutions of a known DNA concentration corresponding to a known number of bacteria for each group or bacterial species. The calculation of the percentage for each bacterial species or group was performed considering the quantit...
Fecal water content
To obtain the fecal water content, 500 mg of each fecal sample was weighed and then lyophilized in a freeze-drier (Christ Alpha 1-2 LD, Osterode am Harz, Germany). Water content was expressed as the percentage of weight loss of stool samples.
Physical activity
When physical activity was compared between groups, there was a significant difference in the percentage of moderate physical activity in the active group (29.96±5.61%) versus the sedentary group (22.82±3.64%) (p = 0.039), while there were no significant differences in light physical activity measurements. The average expenditure was higher in the active group (145±31 kcal/h) than in the sedentary group (105±22 kcal/h), and consequently the total energy expenditure was also higher for active women (3432±911 kcals) than for sedentary women (2523±982 kcals) (p = 0.048). No significant differences were found for the remaining determined variables. Differences in sedentary behavior were also reflected in the variables "maximum time per sedentary break on working days" (SED = 691±96 min vs ACT = 515±91 min; p = 0.005), "average time...
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Evidence quotes (6)
Forty premenopausal women were recruited according to inclusion criteria: age 18-40 years and body mass index (BMI) 20-25 kg/m 2. Criteria for physical exercise were as follows: sedentary women who did not perform the minimal exercise established as healthy by WHO, ie 3 days of exercise per week for 30 minutes at a moderate intensity [ ]; and for active women, those who performed at least 3 hours of physical exercise per week. Exclusion criteria were any kind of pathology, previous gastrointestinal surgery, antibiotics intake during three months prior to the study, smoking, prebiotics, probiotics, vegetarian or vegan, nutritional or ergogenic complements, pregnancy or lactation. All subjects were Caucasian. The Ethics Committee for Clinical Research of the Ramón y Cajal Hospital (Madrid, Spain) approved the study. Written and informed consent was obtained from all participants. The study is registered in Clinicaltrials.gov with the accession number NCT02901912.
Levels of physical activity performed by both groups of women were measured with an ActiSleep V.3.4.2 accelerometer. (Actigraph, Manufacturing Technology Inc., Shalimar, FL). This allowed us to verify in an objective manner the presorting of sedentary and active individuals as it determines body movements in all three axes. Acceleration, energy expenditure, intensity of physical activity and body position was recorded. Results were analyzed with Actilife software (Actilife6, Actigraph). Recordings were performed for 7 days (5 weekdays and 2 weekend days). Participants were instructed to wear the accelerometer on the right wrist all day except when they had a shower or performed pool activities. Data were considered valid when they contained a record of at least five valid days including at least one weekend day, and at least 10 hours of activity were recorded. The time-sampling interval (epoch) was every minute during the 7 days. According to previous reports, a cut-off of <100 counts/minute was chosen to categorize sedentary time, which typically includes activities such as sitting or working quietly (eg, reading or typing on a computer) [ ]. A sedentary bout was defined as 1 o...
Participants were provided with a plastic device to collect stool samples, which were stored at -80°C until extraction. DNA was extracted using the commercial Power Soil DNA Kit (Mobio, CA) with a bead-beating homogenizer (Bullet Blender® Storm, Next Advance, NY). The concentration and purity of DNA was measured using the Quant-iT PicoGreen dsDNA Assay Kit (ThermoFisher Scientific, Waltham, MA).
Quantitative PCR (qPCR) analysis was carried out on a CFX Connect™ Real-Time PCR Detection System (BioRad, Barcelona, Spain) using SYBR Green I chemistry (BioRad) in 20 µL reactions containing one microliter (1-10 ng) of DNA template and 200 nM of primers. Cycling parameters were 95 °C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, 1 minute at the established annealing temperature and 72°C for 45 seconds. Subsequently, melting curve analysis was performed, in which fluorescence was measured as the temperature increased from 50°C to 95°C. For bacterial quantification, standard curves were made using serial dilutions of a known DNA concentration corresponding to a known number of bacteria for each group or bacterial species. The calculation of the percentage for each bacterial species or group was performed considering the quantity of bacteria obtained per gram of stool obtained with a universal primer pair as 100% [ ]. For Bifidobacterium longum and Roseburia hominis detection, primers were manually designed considering the conserved regions in the 16s rRNA gene and the hypervariable regions to avoid cross-reaction with phy...
To obtain the fecal water content, 500 mg of each fecal sample was weighed and then lyophilized in a freeze-drier (Christ Alpha 1-2 LD, Osterode am Harz, Germany). Water content was expressed as the percentage of weight loss of stool samples.
When physical activity was compared between groups, there was a significant difference in the percentage of moderate physical activity in the active group (29.96±5.61%) versus the sedentary group (22.82±3.64%) (p = 0.039), while there were no significant differences in light physical activity measurements. The average expenditure was higher in the active group (145±31 kcal/h) than in the sedentary group (105±22 kcal/h), and consequently the total energy expenditure was also higher for active women (3432±911 kcals) than for sedentary women (2523±982 kcals) (p = 0.048). No significant differences were found for the remaining determined variables. Differences in sedentary behavior were also reflected in the variables "maximum time per sedentary break on working days" (SED = 691±96 min vs ACT = 515±91 min; p = 0.005), "average time per sedentary break on working days" (SED = 81.15±14.71 min vs ACT = 58.82±15.89 min; p = 0.038), and in the variable "total time in sedentary bouts on working days" (SED = 311±21 min vs ACT = 367±35 min; p = 0.027).
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