Subject Recruitment: We directed a cross-sectional population-based study from July 2015 to March 2020. The study was sanctioned by the institutional ethics committee. Study subjects were randomly designated to have approximate representation from each income group (higher ~ 10%, middle ~ 65–70%, and lower ~ 15–20%) from forty-one residential sites in Delhi. Subjects with pregnancy, severe end organ damage or chronic diseases, malignancy, and known T2DM and other endocrine illnesses were excluded from the study. After informed consent, 797 females, 20–60 years of age, were enrolled in this study. The institutional and licensing committee approved all experiments and it is also confirmed that all experiments were performed in accordance with relevant guidelines and regulations.
Demographic and Clinical Profiles: Demographic and clinical profiles, medical history (personal and family), socioeconomic features, skin exposure and time of sunlight exposure were recorded with the use of pre-validated questionnaire. Skin exposure was measured as percentage of body surface area (face/hands, face/hands and arms, and face/hands and legs) exposed to sunlight. The interval of sun exposure (minutes/day) was evaluated in the following mode; < 5 minutes, 5–15 minutes, 15–30 minutes and > 30 minutes. Blood pressure was documented by a standard mercury sphygmomanometer, over the right arm in sitting situation.
Anthropometric Measurements: BMI, circumferences [waist circumference (WC) and hip circumference (HC)] and skinfold thickness at 6 sites (biceps, triceps, anterior axillary, suprailiac, subscapular and lateral thoracic) was recorded (13). Waist-hip ratio (WHR) and waist-height ratio (WhtR) was calculated. Sum of all skinfolds (Σ6SF, total skinfolds), ratios of subscapular and triceps skinfolds (SS/TR ratio), central skinfolds (sum of subscapular and suprailiac) and peripheral skinfolds (sum of biceps and triceps) were also calculated.
Biochemical Analysis: Venous blood sample was collected into vacutainer tubes containing plain and EDTA vials. The fasting blood sample was processed into different aliquots (including whole blood and blood clot) within 2hours into 1.0mL FluidX tubes (FluidX, Cheshire, UK) and frozen at − 80°C freezer (Thermo Fisher Scientific, Waltham, MA, USA).
Fasting blood glucose (FBG) and serum 25-hydroxy vitamin D [25(OH) D] levels were analysed as previously described (13). The intra-assay coefficient of variation of 25 (OH) D was 1.81% and the coefficient of inter-assay was 2.34%.
DNA Isolation and Quantification: DNA was separated from peripheral blood mononuclear cells using the QIAamp DNA extraction kit (Qiagen, Hilden, Germany) and stored at -20oC for the future experiments (14). After DNA isolation, the DNA samples were quantified and diluted to 50 ng/µL. The concentration and quality of DNA were both measured by using a nanodrop (Nanodrop Technologies, Wilmington, NC, USA) and samples included for analysis all had an optical density ratio A 260/A280 > 1.8.
Measurement of Leukocyte Telomerase Length: LTL was analysed with a quantitative polymerase chain reaction (qPCR) based technique that compares telomerase repeat sequence copy number (T) to a reference single copy-gene copy number (S) as previously described (15, 16). The telomerase length for each sample was estimated using the telomerase to single copy gene ratio (T/S ratio) with the calculation of ∆Ct [Ct (telomere)/Ct(single gene)]. T/S ratio for each sample (x) was normalized to the mean T/S ratio of the reference sample [2−(∆Ctx−∆Ctr) = 2−∆∆Ct], which was used for the standard curve, both as a reference sample and as a validation sample. The Measurement consists of determining the relative ratio (T/S ratio) of ng of telomerase (T) to ng of albumin (single-copy gene, S) in experimental samples using a standard curve. The T/S ratio is proportional to the average telomerase length. All qPCR assay was performed using filtered pipette tips to prevent amplification of contaminants. Reactions were set up on ice to prevent DNA polymerase activity, non-specific amplification and to minimize potential primer-dimerization. All analyses were done blinded to cross sectional status of the individual.
The coefficient of variations (CVs) of the inter-plate T/S were 11.6%, and 12.2% for the long and short telomerase QC samples, respectively. Inter- and intra-plate CVs of calibrator DNA samples were 10.2%, and 8.3%, respectively. Mean ratio of long to short telomerase QC samples in our assays was 3.9 with 4.5% CV. All samples in our study were assayed in triplicate, and the results were consistent. Less than 12% of samples had a T/S CV more than 10%.
Overweight and obesity were defined as BMI 23-24.9 kg/m2 and > 25 kg/m2, respectively (17). Abnormal blood pressure was ≥ 130/85 mmHg. Prediabetes was defined as FBG levels ≥ 100 and 125.9 mg/dl. Serum 25(OH) D status was defined as deficient (< 10 ng/ml], insufficient [10.1–30 ng/ml) or sufficient [30.1–100 ng/ml) (18). LTL were categorised in quartiles, 0–25th percentile (1st quartile; LTL ratio < 0.83), 26th − 50th percentile (2nd quartile, LTL ratio 0.84–0.87), 51st − 75th percentile (3rd quartile, LTL ratio 0.88–0.93), and 76th − 100th percentile (4th quartile, LTL ratio 0.94–0.98). Because several values of LTL were gathered around cut-off values of quintiles, slightly changed numbers of subjects were separated in each quintile.
Complete data were entered in an Excel worksheet (Microsoft Corp, Washington, USA). The distribution of demographic, clinical profiles, socioeconomic, medical history (personal and family), behavioral characteristics, sun and skin exposure and biochemical profiles were confirmed for estimated regularity. Mean and standard deviation and number (%) was used to summarize the variables. Relationships between LTL and various indices of body composition were identified by Pearson correlation analyses. Associations with LTL were estimated using partial correlations that adjusted by age. Association of categorical variables were assessed by chi-square/Fisher exact test. The continuous variables were compared between obesity by independent t-test/Wilcoxon rank sum test, as appropriate. The comparisons of clinical, biochemical, anthropometry and body composition profiles among different LTL quartiles were performed by mean of ANOVA and Kruskal-Wallis test, as appropriate, and further trend was seen by non-parametric trend test. Univariate and multivariable linear regression analysis was used to find independent effect of LTL obesity marker after adjusting confounder. Complete data was analysed using Stata − 14 (LLC 4905 Lakeway Drive College Station, Texas 77845 − 4512. USA). For all above, p value of < 0.05 was considered as statistically significant.