Forty-two subjects (aged 18–65 years) with similar dietary habits were recruited from a rural community in Shaanxi, China. We used a standard questionnaire administered by professional staff to collect basic demographic information (age, sex, education, ethnicity, occupation, physical activity, cardiovascular disease-related history, and physical examination findings). Hypertension was defined as systolic BP (SBP) ≥140 mmHg and/or diastolic BP (DBP) ≥90 mmHg, and/ora history of hypertension with current use of anti-hypertensive medications.
The exclusion criteria included: stage 2 hypertension; secondary hypertension; history of cardiovascular disease, chronic liver disease, chronic kidney disease, or diabetes; pregnancy; and alcohol abuse.
This study was performed in accordance with the Declaration of Helsinki and was approved by the ethics committee of the First Affiliated Hospital of Medical School, Xi’an Jiaotong University (Code: 2015-128). All participants provided signed informed consent. The trial registration number was NCT02915315 (http://www.clinicaltrials.gov), with a date of registration of 27September, 2016.
Dietary salt intervention and physical examination
The dietary intervention included a 3-day baseline diet characterized by habitual salt intake, followed by a 7-day low-salt intervention (51.3 mM sodium or 3.0 g NaCl per day), and then a 7-day high-salt intervention (307.8 mM sodium or 18.0 g NaCl per day). Prepacked salt was added to salt-free meals cooked by the study kitchen; participants ate their breakfast, lunch, and supper under the supervision of professional staff during the whole intervention period to ensure compliance with the intervention protocol. Any food that was not provided by study personnel was forbidden. Physical examinations, including height, weight, and waist circumference measurements, were conducted twice on the last day of each period.We measured brachial-ankle artery pulse wave velocity at the same time.
BP measurement and salt sensitivity definition
BP was measured by certified physicians using standard mercury sphygmomanometers according to the protocol recommended by the American Heart Association. Participants were instructed to sit in a resting position for more than 5 minutes after avoiding exercise; smoking; and alcohol, coffee, or tea consumption for at least 30 minutes before BP measurement. BP was measured 3 times at 1-minute intervals on each day of baseline observation and on the last 2 days of the low- and high-salt intervention periods; we recorded the mean value. The BP of each participant was measured by the same physician using the same sphygmomanometer to avoid observation variation. Mean arterial pressure (MAP) was calculated as MAP = (SBP/3) + (DBP × 2/3). Due to the lack of an authoritative consensus on the definition of salt sensitivity based on BP, we classified subjects who demonstrated at least a 10% increase in MAP between the low-salt and high-salt diets as salt-sensitive (SS) and the others as salt-resistant (SR).
Blood samples were obtained by venipuncture on the last morning of each intervention phase. Within 2 hours of collection, staff centrifuged the samples at 3,000 ×g for 10 min to separate EDTA plasma and serum, which were shipped to a central laboratory via standardized procedures where they were stored at −80°C until further analysis. We measured serum creatinine, uric acid, fasting serum glucose, total cholesterol, triglyceride, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol levels using an automatic biochemical analyzer (model 7600; Hitachi, Tokyo, Japan).
We measured plasma S1P levels by liquid chromatography–tandem mass spectrometry. We used a previously described protocol with minor modifications. Plasma (100 μL) was deproteinated by the addition of methanol (400 μL). The internal standard D-erythro-C17-sphingosine-1-phosphate (10 μL, 10 μM; Avanti Polar Lipids, Alabaster, AL, USA) was used to correct for variations in sample preparation and instrument response. We cleared the extracts by centrifugation (5810R Eppendorf, 12,000 rpm, 10 min; Eppendorf, Wesseling-Berzdorf, Germany). We subjected the cleared extracts to reverse-phase chromatography on an Agilent Eclipse XDB C-18 analytical column (2.1 mm × 150 mm, 3.5 μm; Agilent Technologies, Santa Clara, CA, USA) at a flow rate of 0.3 mL/min. S1P was eluted using a ballistic gradient (30% to 85% [v/v] methanol and 0.2% [v/v] formic acid) and measured with a Shimadzu high-performance liquid chromatography system coupled to an API 4000™ tandem mass spectrometer (Applied Biosystems/MDS SCIEX, Framingham, MA, USA). The quantification determination was performed using multiple reaction monitoring with the m/z 380.1 to 264.3 (M + H S1P parent ion). We generated a calibration curve (0.125–5 μM S1P) to calculate the S1P levels in the samples. The same sample was tested multiple times with a relative standard deviation <5% within 12 hours and <10% within 6 days when stored at room temperature.
24-hour urinary sodium and potassium determination
We collected 24-hour urine samples on the last day of each period, which we froze at −40°C until further analysis. We determined the urinary concentrations of sodium and potassium using ion-selective electrodes (Hitachi, Tokyo, Japan). The 24-hour urinary excretion of sodium and potassium were calculated as 24-hour excretion = [Na+ or K+] × 24-hour total urine volume.
We performed statistical analyses with SPSS Statistics 22.0 (IBM, Chicago, IL, USA). Continuous data are presented as the mean ± standard deviation. Categorical data are shown as frequency and percentage. Differences in repeated measures were analyzed by repeated-measures analysis of variance. Differences in characteristics between the SS and SR groups were analyzedby independent t-tests for continuous variables, where appropriate; they were otherwise analyzed by Mann–Whitney U test.We determined correlations by calculating Pearson’s correlation coefficient, where the residuals were normally distributed, and by calculating Spearman’s correlation coefficient in other cases. A two-tailed P-value ≤0.05 was considered statistically significant.