The YHIP study, from which the blood samples are taken, was a prospective three-arm single-center study of the effects of two types of yoga on BP and quality of life . There were two intervention groups and one control group. The study was designed as a matched controlled open clinical trial. Blood tests, BMI-, waist circumference-, BP measurements and assessments of lifestyle, health status and quality of life  were carried out at baseline and after 12 weeks of intervention. The groups were matched based on SBP after baseline assessments.
Patients and recruitment
In January 2011, adult patients with diagnosed hypertension were identified by electronic charts search at Svedala Health Care Center in Southern Sweden. Patients were invited to participate if their BP when most recently measured at the health care center was between 120 and 160/80 and 100 mm Hg (e.g. normal, high normal and grade 1 hypertension levels). The patients who agreed to participate were invited to the health care center for baseline assessments. Patients with BP values of 120–179/<110 mmHg in the baseline check were eligible for enrollment. Fasting blood samples were collected and analyzed for HbA1c, FP-glucose, cholesterol, TGs, HDL, LDL and hs-CRP. For each patient three cryo tubes with blood were frozen for future analysis of IL-6 and IL-10. Baseline assessments and study questionnaires (lifestyle and health status survey and quality of life were completed after written informed consent was obtained from the participants.
All participants were requested not to change their medication during the study, and any change in medication was registered at follow-up after 12 weeks. For further details regarding patients and procedures please refer to our previously published article .
The yoga practiced in the YHIP study is a form of Kundalini yoga developed at the Institute for Medical Yoga (IMY) in Stockholm, Sweden . Intervention group 1 (28 persons) was divided into three smaller groups, each consisting of 8–12 participants. Each group met once a week for 60 min at the health care center to practice yoga with a yoga instructor. The participants were encouraged to practice yoga 30 min every day at home between the yoga classes. The participants in intervention group 2 (28 persons) were each given a doctor’s appointment (20 min) during which they received instructions for two yoga exercises to perform at home for a combined total of 15 min a day. No changes were made for the participants in the control group, who received treatment as usual. In order to evaluate compliance with yoga practice, each participant received a yoga calendar in which to record when they did yoga. For further details regarding the interventions please refer to our previously published article .
The main outcome measure was change in blood inflammatory and metabolic factor levels. We chose to analyze inflammatory and metabolic factors with known or suspected connections to CVD.
Blood samples were collected at baseline and follow-up for assessment of the following factors: HbA1c, FP-glucose, cholesterol, TGs, HDL, LDL and hs-CRP. The blood samples were drawn in the morning after a fast since midnight.
HbA1c was analyzed using the Bio-Rad Variant II chromatographic method (reference range 31–44 mmol/mol) by Swedish Mono-S high-performance ion-exchange liquid chromatography. Due to an instrument change at the laboratory during the intervention period, the following were analyzed by different methods at baseline and follow-up: FP-glucose, cholesterol, TGs and HDL. At baseline, they were measured in plasma using an LX20 analyzer (Beckman Coulter Inc., Brea, CA). At follow-up the above samples were analyzed using a cobas 6000 Analyzer (Roche Diagnostics, SA). LDL level was calculated using the Friedewald formula . The results from follow-up were recalculated according to regression equations provided by the laboratory to compensate for any differences due to the change in instrument.
The blood samples from baseline and follow-up were used for IL-6 and IL-10 levels. Serum was isolated from blood by centrifugation at 10,000 g at 4 °C for 10 min. Samples were analyzed using Bio-Plex Pro human cytokines assay (Bio-Rad Inc., Hercules, CA), according to the manufacturer’s instructions with a few modifications. Briefly, samples were diluted 1:3 in the sample diluent provided with the kit and incubated with magnetic beads coupled to specific antibodies. IL-6 and IL-10 were detected with premixed detection antibody. Beads were read on a Bio-Plex Suspension Array System and the data were analyzed using Bio-Plex Manager™ software (version 4.0). Absolute concentrations were measured from a standard curve generated from nine serially diluted standards provided with the kit. Each sample was analyzed in duplicate. Values are presented in pg/mL. Each run included controls of known concentration for each cytokine and a blank.
The health status and lifestyle survey was designed for this study and is not validated. The survey contains questions regarding comorbidity for diabetes and cardiovascular disease, smoking and drinking habits and physical activity.
The participants were instructed to mark the dates they completed the yoga training. At follow-up, they submitted their yoga calendars. The information in the calendars was not controlled or questioned.
BP was measured in a standardized way, in a sitting position after 5–10 min of rest with validated electronic BP devices. The mean of two readings were calculated (mean of three when the first and second reading differed by >5 mm Hg). The measurements were carried out by trained nurses and care assistants.
Assuming a mean treatment difference in SBP of 5 mmHg between the yoga at home and control groups, a standard deviation of 6 mmHg and a drop-out rate of 30 %, 33 patients per group would provide 80 % power to detect a statistically significant difference at the 5 % level using a two-sided test.
One-way-ANOVA was used to determine whether there were any significant differences in baseline data between the groups. For the laboratory results that were not normally distributed (FP-glucose, HbA1c, hs-CRP, IL-10 and IL-6) we used the Kruskal-Wallis test instead.
Differences in blood test parameters, BP and waist circumference between baseline and follow-up in each group were calculated by paired-samples Student’s t-test. Differences in mean change between the yoga groups and the control group were calculated by independent-samples Student’s t-test. For the laboratory results that were not normally distributed (FP-glucose, HbA1c, hs-CRP, IL-6 and IL-10), the differences within and between groups were calculated by Wilcoxon test and Mann–Whitney U-test, respectively.
Version 22 of the IBM SPSS Statistics was used for the statistical analysis.
All patients who attended follow-up appointments were included in the analyses (as observed cases, OC). We also made calculations in which patients who did not perform yoga in 9/12 weeks or who changed their medication were excluded (per-protocol set, PPS). This criterion (9/12 weeks) was set up together with the IMY founder, and it was not known to the patients.
The study conforms to the principles outlined in the Declaration of Helsinki and was approved by the Regional Ethical Review Board in Lund, Sweden (2010/728). The study was registered at ClinicalTrials.gov (NCT01302535).