Subjects (Table.1). Ninety-nine subjects were recruited from the University of the Third Age (U3A) (Fig. 1). The age of 60–90 years was the inclusion criterium. The exclusion criteria included: any experience in competitive sport, acute infectious diseases, oncologic diseases, neurodegenerative diseases, and an implemented pacemaker, based on the assessment of the responsible physician and the investigator. The current health status and lifestyle of the subjects were estimated by means of the health history questionnaire [18]. Eventually, the study included fifty-four subjects (females n = 47 males n = 7), aged 65–88 years, who represented the successful ageing according to the definition by Geard et al. [19]. On the basis of the assessment of functional and cardiorespiratory fitness, according to Åstrand [20], as well as the gait speed measurement, according to Middleton et al. [21] and Studenski [22], thirty-four subjects (females n = 31, males n = 3) were included in the physically active group whereas the inactive group was composed of twenty subjects (females n = 16, males n = 4). All U3A students were informed of the aim of the study and gave their written consent for participation in the project. The protocol of the study was approved by The Bioethics Commission at Regional Medical Chamber Zielona Gora, Poland (No01/66/2017, No21/103/2018) in accordance with the Helsinki Declaration.
Table 1
Anthropometrics and body composition in the elderly (mean ± SD).
|
Active
n = 34
|
Inactive
n = 20
|
Active vs. Inactive p level
|
η2
|
Age [yr.]
|
70.2 ± 5.8
|
73.5 ± 5.4
|
< 0.05
|
0.008
|
Weight [kg]
|
69.8 ± 11.8
|
67.1 ± 11.3
|
0.567
|
0.000
|
Height [cm]
|
160.3 ± 6.0
|
159.7 ± 7.2
|
0.573
|
0.040
|
BMI [kg/m2]
|
27.1 ± 3.6
|
26.3 ± 4.1
|
0.529
|
0.006
|
FM [kg]
|
24.1 ± 5.8
|
22.0 ± 5.9
|
0.268
|
0.004
|
FM%
|
34.3 ± 4.7
|
32.7 ± 6.2
|
0.348
|
0.006
|
FFM [kg]
|
45.7 ± 7.7
|
45.1 ± 8.7
|
0.622
|
0.012
|
SBP [mmHg]
|
145.1 ± 19.2
|
151.3 ± 20.9
|
0.264
|
0.017
|
DBP [mmHg]
|
81.2 ± 11.6
|
79.2 ± 12.9
|
0.602
|
0.004
|
6MWT [m]
|
527 ± 52
|
388 ± 59
|
< 0.001
|
0.662
|
Gate speed [m/s]
|
1.5 ± 0.1
|
1.0 ± 0.1
|
< 0.001
|
0.662
|
VO2max [mL/kg/min]
|
35.8 ± 5.7
|
32.8 ± 4.0
|
0.131
|
0.060
|
Abbreviations: BMI Body Mass Index, FM Fat Mass, FFM Fat-Free Mass, SBP systolic blood pressure, DBP diastolic blood pressure, 6MWT 6-min walking test, VO2max maximal oxygen consumption. The measurements in groups are compared by the one-way ANOVA or the Manna-Whitney non-parametric test (if the normality assumption is violated). |
Body composition. Body mass (BM) and body composition fat-free mass (FFM) and FM were estimated by a bioelectrical impedance method using Tanita Body Composition Analyser MC-980 (Japan) calibrated prior to each test session in accordance with the manufacturer’s guidelines. Duplicate measures were taken with the participant in a standing position; the average value was used for the final analysis. The recurrence of measurement was 98%. The measurements were taken between 7:00 and 9:00 a.m., before blood sampling.
Functional fitness. The 6-min walking test (6MWT) was performed according to technical standards of European Respiratory Society and American Thoracic Society [23]. A marked walkway was laid out in a 50-m rectangular area (dimensions: 20 × 5 m), with cones placed at regular intervals to indicate the distance covered. The aim of the test was to walk as quickly and as far as possible over a span of six minutes. The subjects were allowed to self-pace (a preliminary trial was useful to practice pacing) and rest as needed. The total distance walked in the test was recorded and the 6MWT gait speed was then calculated by the following equation: 6MWT gait speed (m/s) = total distance(m)/360 s. The gait speed ranging from 1.3 to 1.8 m/s classified the older adults as active and the gait speed < 1.3 m/s classified them as inactive according to Middleton et al. [21].
Cardiorespiratory fitness. The measurement of maximal oxygen consumption (VO2max) was performed via the indirect method known as the Åstrand-Ryhming bike test (6-min submaximal exercise test) which relies on the linear relationship between heart rate (HR) and VO2 to predict maxVO2 and which is recommended for both men and women of various ages [20]. Each subject performed a 6-min submaximal exercise test on a cycle ergometer eBike GE Healthcare (Germany). Initially, the study subjects rested for 15 minutes prior to the measurement of their resting HR. The seat height and handlebars were adjusted for each subject prior to the test. According to normative data for submaximal exercise test, the subjects who reached the values of VO2max > 35 mL/kg/min were classified as active (high activity level) and the remaining ones were determined as inactive (average and low activity level).
Type and amount of physical activity. The type and weekly amount of physical activity was evaluated by Community Healthy Activities Model Program for Seniors (CHAMPS) [24]. The CHAMPS was originally designed to assess the types and intensity levels of physical activity including lighter (e.g. leisurely walking, water gymnastics, stretching, Tai-Chi) as well as more vigorous activities (e.g. dancing, cycling, swimming). Currently, the CHAMPS also includes a group of items related to a sedentary lifestyle e.g. sitting and chatting with friends.
Blood sampling. Blood samples were taken from the median cubital vein using S-Monovette-EDTA K2 tubes (Sarstedt, Austria) for flow cytometry analysis and morphology and S-Monovette - serum tubes were used for other biochemical markers. Serum samples were left to clot for 45 min before centrifugation and then centrifuged at 3000 g and + 8oC for 10 min. Aliquots of serum were stored at -80 °C.
Flow cytometry analysis. Cytometric analysis was performed using eight-parameter CyFlow Space Sorter flow cytometer by Sysmex Partec (Germany). For the analysis of immune cells, CyLyse kit by Sysmex (Germany) was used. 100 µl venous blood was mixed with fluorochrome labeled monoclonal antibodies (CD8 APC, CD4 FITC, CD45 RA Pacific Blue™ CD45RO PE) and incubated for 15 minutes in the dark at room temperature. After the incubation 100 µL of Leukocyte Fixation Reagent A was added and incubated again in the dark for 10 minutes. In the last step, 2.5 ml Erythrocytes Lysing Reagent B was added, mixed and incubated in the dark for 20 minutes and further measurements were made. T helper and cytotoxic lymphocytes were gated by positive surface staining for CD4 and CD8 and were expressed as a percentage of gated lymphocytes. Memory and naïve subpopulations were gated by positive surface staining for CD45RO and CD45RA, respectively. The ratios of CD4+naïve to CD4+memory and CD8+naïve to CD8+memory, as prognostic markers of chronic diseases, were calculated according to Hang et al. [25]. Moreover, the CD4/CD8 ratio was calculated according to McBride and Striker [26] to express the Immune Risk Profile (IRP) associated with altered immune function. The reference values for IRP were adopted from Strindhall et al. study [27]. The ratios ≥ 1 or ≤ 2.5 are generally considered normal, however, a wide heterogeneity exists because of sex, age, ethnicity, genetics, environmental exposures and infections. The high or inverted CD4/CD8 ratio (< 1 or > 2.5) is regarded as an immune risk phenotype and is associated with immunosenescence and chronic inflammatory diseases [27].
Haematological variables. Peripheral blood morphology: white blood cell count (WBC), granulocytes (%GRA), lymphocytes (%LYM), mid absolute count (%MID), red blood cells count (RBC), haemoglobin (HB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets (PLT) were determined by using 3 diff BM HEM3 Biomaxima (Poland).
Biochemical markers. Total cholesterol (TC), high-density lipoproteins (HDL), low-density lipoproteins (LDL), triglycerides (TG) were determined by using BM200 Biomaxima (Poland). The non-HDL cholesterol was calculated by subtracting HDL from the total cholesterol concentration. The serum C-reactive protein (CRP) level was determined in duplicate by DRG ELISA kit (Poland). 0.001 mg/L was established as the detection limit and CV for the CRP kit was set at < 3%. The serum glucose was measured spectrophotometrically using Diaglobal spectrophotometer (Germany).
Statistical analysis. Statistical analyses were performed using the R system, version 3.6.1 [28]. The assumptions for the use of parametric or non-parametric tests were checked using the Shapiro-Wilk and the Levene tests to evaluate the normality of the distributions and the homogeneity of variances, respectively. The significant differences in mean values between the groups (Active vs. Inactive) were assessed by the one-way ANOVA. If the normality and homogeneity assumptions were violated, the Manna-Whitney non-parametric test was used. Additionally, eta-squared (η2) was used as a measure of effect size which is indicated as having no effect if 0 ≤ η2 < 0.01, a minimum effect if 0.01 ≤ η2 < 0.06, a moderate effect if 0.06 ≤ η2 < 0.14, and a strong effect if η2 ≥ 0.14 [29, 30]. Pearson's correlation coefficients were calculated to describe the relationships between a physical activity level and immune cells counts. Statistical significance was set at p < 0.05.