Sodium-24

Our primary outcome was the difference between the intervention and the control group in the change of salt intake as measured by 24 hour urinary sodium from baseline to the end of the trial. The secondary outcome was the difference between the two groups in the change of blood pressure.
All outcome assessments were carried out at baseline and at the end of the trial in exactly the same way in all schools for all participants, irrespective of their assignment to intervention or control group.
We carried out two consecutive 24 hour urine collections. Trained research staff carefully instructed participants on how to accurately collect 24 hour urine samples. On the first visit to the participants’ home, the researchers asked the participants to empty their bladder and discard the urine. The researchers recorded the start time and date of the 24 hour urine collection. They then gave the participants the collection equipment including containers and collection aids such as carrier bags. The participants were instructed to collect all subsequent urine voids over the next 24 hour period. On the second day at the same time, the researchers revisited the participants’ home and asked them to pass the last urine into the container. The researchers recorded the finish time of the first 24 hour urine collection. The researchers then gave the participants the collection equipment for the second 24 hour urine collection and repeated the process. Participants were told to take spare urine containers with them when they went to school or work. Spare collection equipment was also available in the schools, in case children forgot to bring containers. For most families, collections were made on the same days of the week for baseline and follow-up. In the event that the participant missed one or more urine voids or spilt >10% of the total 24 hour urine volume, the participant was asked to do a further 24 hour collection.
The urine samples were measured for volume and sodium, potassium, and creatinine concentrations. An ion selective electrode method was used for sodium and potassium analysis (AC9102 electrolyte analyzer, Audicom Medical Technology, Jiangsu) and Jaffe method for creatinine (Hitachi 7080 automatic biochemical analyzer, Japan). The biochemists who performed the urinary electrolyte measurements were not aware of the participants’ group allocation.
We used the average of the two 24 hour urinary measurements at each time point in the analysis. In one child and six adults, however, we had only one 24 hour urine collection at baseline; and in one adult we had only one 24 hour urine collection at follow-up. In these cases, we used one 24 hour urinary measurement.
Trained researchers measured blood pressure and pulse rate at the participants’ homes using a validated automatic blood pressure monitor (Omron HEM-7301-IT, Amsterdam) with an appropriately sized cuff. After participants had rested for 10 minutes in a quiet room, three readings were taken in the right arm at two minute intervals with the participants in the sitting position and the arm supported at heart level. We used the average of the last two measurements for the analysis. Body weight and height were measured in participants without shoes or heavy clothes, with a standardised protocol. Both indoor and outdoor temperatures were measured with a thermometer (Anymetre, JR913).

Blood was collected 24 h before surgery on sodium citrate from 24 patients (male, aged 69.6 ± 8.7 years, range 60–82) with degenerative AAA,^{20 (link)} and 24 healthy male controls (aged 68.7 ± 8.5 years, range 58–78) examined at ‘Centre d’Investigation Preventative et Clinique’ (IPC, Paris).^{23 (link)} A significant ILT was present in all the AAA studied. Circulating aortic channels were measured at the level of maximal dilatation on CT scan. ILT proportion was calculated as followed: (maximal aortic diameter−circulating channel)/maximal aortic diameter and was expressed as percentage. The mean maximal AAA diameter was 54.0 ± 8.55 mm. The mean ILT thickness was 25.5 ± 8.4 mm, representing 46.8 ± 11.8% of the AAA dilatation.
Samples of AAA thrombus and residual wall were obtained during AAA surgical repair from these 24 and five additional patients. ILT (n = 29) were dissected into luminal, intermediate, and abluminal layers^{9 (link),20 (link)} and the wall was separated into media and adventitia (n = 10). The thrombus layers, media, and adventitia were cut into small pieces (1 mm³) and separately incubated in RPMI-1640 medium (Gibco) for 24 h at 37°C (2 ml/g wet tissue). The tissue-culture media containing released material were then collected and stored at −80°C after determining the protein concentration by the Bradford assay (Bio-Rad). Ethics Committee advice and patient and healthy volunteer informed consent were obtained (RESAA and AMETHYST studies, CPP Paris-Cochin no 2095, 1930, and 1931). The investigation conforms with the principles outlined in the Declaration of Helsinki.
In vitro serum from three healthy volunteers was obtained 2 h and 1, 2, 3, 4, and 7 days after clotting of blood at 37°C. Paired citrated plasma samples were used directly or recalcified (3×10⁻² M CaCl₂) to induce fibrin formation in the absence of blood cells. Sera, plasma, and recalcified plasma were then kept at −80°C until ELISAs were performed.