1. Methods to measure salt intake at individual level: gold standard, proxy and calibration needs Francesco P Cappuccio MD MSc DSc FRCP FFPH FBHS FAHA Professor of Cardiovascular Medicine & Epidemiology Head, WHO Collaborating Centre University of Warwick, Coventry, UK Disclosures: Technical Advisor to the World Health Organization, the Pan American Health Organization, Member of C.A.S.H., W.A.S.H., UK Health Forum and Trustee of the Student Heart Health Trust Vice-President, British Hypertension Society – all unpaid.

2. How much sodium are we consuming?

3. Methods to estimate sodium intake Duplicate diets Dietary surveys – Food frequency – 7-day weighed records – Food diary – 24-hour recall Urinary collection – Spot (casual) – Overnight – 24-hour Norfolk-EPIC Study (Khaw et al. 2004) Men Women

4. Background In steady state, most of Na eaten in a day is excreted through urine in the following 24-h. 24-h urinary Na excretion ‘gold standard’ for measuring sodium (salt) intake both in individuals and in populations. 24-h collections often deemed inconvenient, hence alternative methods suggested (e.g. spot and timed urines). Their reliability and reproducibility often disappointing. However, in recent years a confusion has arisen as to what extent ‘proxy’ measures can be used to assess ‘salt’ intake. ‘Population’ estimates (to monitor population policies) vs ‘Individual’ estimate (to use as exposure in research studies).

5. Background Gold standard: 24h urine collection High participation burden Completeness Need to explore alternatives – Casual spot – Timed (Day, Eve, Night) – Estimation v Extrapolation – Individual v Group Their reliability and reproducibility often disappointing

6. “Timed” urines Less participant burden than 24 hour collections More variable at individual level but may give good estimate of group mean Desirable alternative for monitoring program effects over time (validation necessary) Need baseline 24 hour urine assays to compare between populations or time points Role of urinary creatinine Should learn from monitoring program for iodisation Could be used for ‘effective’ monitoring of iodine (ideally in adult populations)

7. ‘Spot’ urines Less participant burden than 24 hour collections Highly variable at individual level but may give estimate of group mean Less desirable for monitoring program effects over time Highly dependent on hydration, volume, residual bladder volume Currently used for monitoring iodine (mainly children and women of childbearing age) Extrapolated to 24h (validation studies published – interpretations vary - population specific ?)

8. Overnight collections Less participant burden than 24 hour collections May give biased estimates of sodium excretion – (greater % excretion overnight in hypertensive individuals…) Undesirable for monitoring program effects over time

10. Measurement of sodium intake Sodium intake highly variable day to day  Misclassification of individual intake and increase in variance of group (mean unbiased) Between individuals Within individuals 2B2B2B2B 2W2W2W2W Reliability =   2 B  2 B +   2 W Liu K et al. Hypertension 1979; 1: 529-36

11. The variability of 24-hour urinary sodium (…) was studied in a sample of 22 Neapolitan men with mild blood pressure elevation. On 5 days within a 1-month period, 24-hour urine specimens were collected by each subject. The estimated ratio of intraindividual-to- interindividual variance was 1.12 for urinary sodium, (…). Based on these values, five 24-hour urine collections are necessary to reduce to less than 10% the diminution of the correlation coefficient between urinary sodium and another related variable; this number is substantially lower than that found in previous studies in a North American population sample, but similar to the one reported for Chinese population samples (…). Siani A et al. Hypertension 1989; 13: 38-42 How many 24h collections are needed to characterize an individual’s sodium excretion?

12. We performed 2 independent ultralong-term salt balance studies lasting 105 (4 men) and 205 (6 men) days in 10 men We controlled dietary intake of all constituents for months at salt intakes of 12, 9, and 6 g/d and collected all urine. Urinary recovery of dietary salt was 92% of recorded intake. Even at fixed salt intake, 24-hour urine collection for sodium excretion (UNaV) showed infradian rhythmicity. Collecting seven 24-hour urines and sodium intake samples improved classification accuracy to 92%. Single 24-hour urine collections at intakes ranging from 6 to 12 g salt per day were not suitable to detect a 3-g difference in individual salt intake. Repeated measurements of 24-hour UNaV improve precision. This knowledge could be relevant to patient care and the conduct of intervention trials. Lerchl K et al. Hypertension. 2015; 66: 850-7

13. Lerchl K et al. Hypertension 2015; 66: 850-7 Multiple collections reduce the variability and improve the predictive value

14. Data Study 1: Discovery sample 915 British men & women aged 40-59 (297 W, 326 B, 292 SA) Study 1: Discovery sample 915 British men & women aged 40-59 (297 W, 326 B, 292 SA) Study 2: Validation sample 148 Italian White men aged 32- 75 Study 2: Validation sample 148 Italian White men aged 32- 75 Na, Cr in 24-h and timed spot urines Reliability and Reproducibility Tanaka’s method † Arithmetic extrapolation method Reliability and Reproducibility Tanaka’s method † Arithmetic extrapolation method †: Tanaka T et al. J Hum Hyper,2002;16, 97-103. Ji C et al. Nutr Metab Cardiovasc Dis 2014; 24: 140-7

15. Mean diff 95% CI Overestimation Underestimation Bland-Altman plot comparing estimated 24h UNa by Tanaka’s method and actually measured 24h UNa Bland-Altman plot comparing estimated 24h UNa by Tanaka’s method and actually measured 24h UNa Ji C et al. Nutr Metab Cardiovasc Dis 2014; 24: 140-7

16. Bland-Altman plot Tanaka’s method Ji C et al. Nutr Metab Cardiovasc Dis 2014; 24: 140-7

17. The Prospective Urban Rural Epidemiological (PURE) Study

18. Peng Y et al. PLoS ONE 2016; DOI: 10.1371/journal.pone.0149655 To assess the validity of three methods (Kawasaki, INTERSALT, Tanaka) for estimating 24h urinary sodium excretion using spot urine samples against measured 24h urinary sodium excretion in a Chinese sample. Sub-study of the PURE study (120 participants 35-70 yrs; 4 excluded – final n=116) Morning fasting time urine and 24h urine Mean bias for the Kawasaki method was the lowest among the three methods, that for the INTERSALT method was the highest. Bland-Altman plots indicated that all three methods underestimated 24-h urinary sodium excretion. Among the three methods, the Kawasaki method was least biased, but was still relatively inaccurate. A more accurate method is needed to estimate the 24-h urinary sodium excretion from spot urine. The Prospective Urban Rural Epidemiological (PURE) Study: SUMMARY

19. Peng Y et al. PLoS ONE 2016; DOI: 10.1371/journal.pone.0149655

20. The PURE Study We compared estimates of 24-h sodium excretion from a single morning fasting urine (MFU) using three different formulae in healthy individuals. We studied 1083 individuals aged 35-70 years from the general population in 11 countries. A 24-h urine and MFU specimen were obtained from each individual. A subset of 448 individuals repeated the measures after 30-90 days. The Kawasaki, Tanaka, and INTERSALT formulae were used to estimate urinary excretion from a MFU specimen. The intraclass correlation coefficient (ICC) between estimated and measured sodium excretion was higher with Kawasaki compared with INTERSALT and Tanaka formulae (P <0.001). The degree of bias (vs. the 24-h urine) for sodium was smaller with Kawasaki compared with INTERSALT and Tanaka formulae (P <0.001 and P = 0.02, respectively). In a diverse population, the Kawasaki formula is the most valid and least biased method of estimating 24-h sodium excretion from a single MFU and is suitable for population studies. Mente A et al. J Hypertens 2014; 32: 1005-14 ‘First void’ (overnight) urine sample Kawasaki equation designed to assess ‘second’ void morning samples Very high rate of incomplete collections (>50%) Bias described higher at higher levels of 24h urinary sodium, likely due to incompleteness Campbell N. J Hypertens 2014; 32: 2499-503

21. Mean bias in predicted minus measured 24h urinary sodium excretion on the same day by prediction model and time of day* Cogswell ME et al. Am J Clin Nutr 2013; 98: 1502-13 * Timed urine samples not independent of 24h sample

22. Cogswell ME et al. Am J Clin Nutr 2013; 98: 1502-13 Morning* Afternoon* Evening*Overnight* Bland-Altman plots of the relative bias between measured and predicted 24h urinary sodium excretion based on INTERSALT equation * Timed urine samples not independent of 24h sample

23. Spot timed urinary Na does not provide reliable and reproducible estimates of 24-h urinary Na excretion in an individual. Spot urines should not be used in cohort studies to estimate individuals’ exposures 24-h urinary collection for the measurement of urinary Na excretion remains the preferred tool for assessing salt intake. Multiple collections are necessary to increase the prediction. Conclusions

24. 1.24-hour urine is the most accurate method for measuring population salt intake if carried out properly and measures are taken to ensure that the 24-hour urine collections are complete. Experts with experience in 24 hour urine collection e.g. endocrinologists, should be engaged to co- ordinate the surveys. 2.If 24-hour urine collection is not feasible, there is some evidence that in a generally healthy adult (18-69) population, spot urine could approximate mean sodium intake and identify populations where salt intake is above the 5gram target. It is noted that this is a subject of ongoing research. 3.To detect change over time in population salt intake, 24-hour urine is the most accurate method for repeat measurement but if not feasible, spot urine could approximate the mean sodium intake with some significant degree of underestimation of the change over time to take into consideration 4.24-hour urine is the most accurate method of assessing individual sodium intake and single spot or short duration timed urine samples are inappropriate to use for this purpose ………………………………………………………