No Evidence That the Skeletal Non-Response to Potassium Alkali Supplements in Healthy Postmenopausal Women Depends on Blood Pressure Or Sodium Chloride Intake

ORIGINAL ARTICLE No evidence that the skeletal non-response to potassium alkali supplements in healthy postmenopausal women depends on blood pressure or sodium chloride intake

LA Frassetto1,4, AC Hardcastle2,4, A Sebastian1, L Aucott2, WD Fraser3, DM Reid1 and HM Macdonald2

BACKGROUND/OBJECTIVES: In vitro studies demonstrate that bone is degraded in an acidic environment due to chemical reactions and through effects on bone cells. Clinical evidence is insufﬁcient to unequivocally resolve whether the diet net acid or base load bone affects breakdown in humans. Increasing dietary salt (sodium chloride, NaCl) mildly increases blood acidity in humans and in rats with increased sensitivity to the blood pressure effects of salt, whereas increased potassium (K) intake can decrease blood pressure. Blood pressure responses to NaCl or K may potentially be a marker for increased bone turnover or lower bone mineral density (BMD) in women at higher risk for osteoporosis and fracture. SUBJECTS/METHODS: We retrospectively analysed data from two data sets (California and NE Scotland) of postmenopausal women (n ¼ 266) enrolled in long-term randomized, placebo-controlled studies of the effects of administration of low- or high-dose dietary K alkali supplementation on bone turnover in relation to sodium or chloride excretion (a marker of dietary salt intake). Mean arterial pressure (MAP) was calculated from blood pressure measures, MAP was divided into tertiles and its inﬂuence on the effect of dietary NaCl and K alkali supplementation on deoxypyridinoline markers of bone resorption and BMD by DEXA was tested. Data was analysed for each data set separately and then combined. RESULTS: Percentage change in BMD after 24 months was less for California compared with North East Scotland (hip: À 0.6±2.8% and À 1.5±2.4%, respectively (P ¼ 0.027); spine: À 0.5±3.4% and À 2.6±3.5%, (Po0.001). We found no effect of dietary alkali treatment on BMD change or bone resorption for either centre. Adjusting for the possible calcium- or potassium-lowering effects on blood pressure did not alter the results. CONCLUSIONS: Blood pressure responses to Na, Cl or K intake did not help predict a BMD response to diet alkali therapy.

European Journal of Clinical Nutrition (2012) 66, 1315–1322; doi:10.1038/ejcn.2012.151; published online 24 October 2012 Keywords: osteoporosis; blood pressure; diet; potassium; sodium chloride

INTRODUCTION acids in frozen 2-h urine specimens did not show an association Dietary factors affecting osteoporosis with BMD or fractures10 and a 2-year intervention study demonstrated no improvement in BMD in subjects treated with Although many factors that affect bone mineral density (BMD) are 11 known, they account for only half of the variability among subjects dietary alkali. when assessing risks for osteoporosis.1 Whether or not blood pressure responses to alterations in dietary acid (from table salt, Salt intake and bone effects NaCl) or base from potassium alkali salts is associated with osteoporosis is unclear.2 Dietary salt may affect bone metabolism via a number of mechanisms. In healthy men, increasing Na intake from 10 to 1500 mmol (0.5–65 g/day) on a fixed calcium intake increased the Dietary acid and bone effects filtered load of calcium and fractional calcium excretion Increases in blood acid loads, marked by increased urinary acid (Po0.001).12,13 Studies suggest that urinary Ca decreases by 0.5 excretion, increase calcium efflux from bone in in vitro;3,4 in to 1.5 mmol (20–60 mg/day) for every 100 mmol (2300 mg) human clinical trials, faecal calcium balance was not significantly decrease in sodium ingested.14,15 In the DASH-Na diet, low salt changed, whereas urine calcium excretion increased,5,6 leading intake was associated with decreased urinary Ca excretion and some authors to suggest that the calcium was coming from significantly reduced bone turnover markers; calcium balance, a bone.4,6,7 In rats and young men, very high dietary NaCl intake more robust indicator of bone health, was not measured.16 (above the usual diet levels) induce lower blood pH and Increases in either intravenous or dietary Na intake are linked to bicarbonate and higher urinary net acid excretion8,9 and are increases in hormones such as parathyroid hormone (PTH)17 that associated with increased urinary markers of bone breakdown.9 are known to affect bone turnover. Increases in dietary chloride However, a recent cohort study that retrospectively measured net intake is associated with decreasing blood pH and serum

1Department of Medicine and Clinical Research Centre, University of California San Francisco, San Francisco, CA, USA; 2University of Aberdeen, Foresterhill, Aberdeen, UK and 3Norwich Medical School, University of East Anglia, Norwich, UK. Correspondence: Dr LA Frassetto, Department of Medicine and Clinical Research Centre, University of California San Francisco, 505 Parnassus Ave, room M1201/campus box 0126, San Francisco, CA 94143-0126, USA. E-mail: [email protected] 4These authors contributed equally to this work. Received 8 December 2011; revised 10 September 2012; accepted 11 September 2012; published online 24 October 2012 Blood pressure, salt and bone LA Frassetto et al 1316 18 bicarbonate (HCO3), leading to higher blood acid levels. Dietary intake and anthropometric assessments. The European Prospective Together, increased Na and Cl intake are associated with Study into Cancer (EPIC) food diary was completed for 4 days before increased urine calcium excretion and increased blood acid randomisation and at 1 year34,35,36 and analysed using ‘Windiets’ (Robert levels, respectively, within the range of normal, and may Gordon University, Aberdeen, UK). Women were weighed 6-monthly on potentially contribute directly or indirectly to bone turnover. balance scales (Seca, Hamburg, Germany) and height was measured at baseline and 2 years with a stadiometer (Holtain Ltd, Crymych, UK). Blood pressure was measured with an automatic sphygmomanometer Salt intake, blood pressure, nitric oxide (NO) and bone (Omron 705CP; Omron Healthcare Ltd, Milton Keynes, UK) and measured 19,20,21 in the same arm at each visit after the patient was settled and before NaCl intake can increase blood pressure in some individuals venipuncture was performed. and potassium intake can lower blood pressure.22 Blood pressure effects in cardiovascular disease have been well studied;22,23 more Markers of bone health. Urine free deoxypyridinoline cross-links (fDPD) recently, factors important in cardiovascular disease are now also 24,25 were measured in 2 h early morning fasted urine samples at baseline, 3, 6, being seen as possibly affecting bone health. 12, 18 and 24 months. Creatinine (Cr) and Na were measured with standard Asymmetric dimethylarginine (ADMA) is a competitive inhibitor automated techniques on a P module (Roche, Lewes, UK), and the results of endothelial NO synthase; increases in ADMA inhibit NO-related are expressed as fDPD/Cr (nmol/mmol). The inter-assay CV for the marker vasodilation, leading to increased blood pressure.26 Fang et al.27 in method was o5.5% across the working range. The method used is a cross-over studies in normotensive rural Chinese demonstrated modification of the HPLC technique published previously utilising mass 37 significantly higher mean blood pressures and ADMA levels in 13 spectrometry (MS) detection of DPD. BMD was measured at the spine salt-sensitive (SS) normotensive subjects on a high salt diet and hip at baseline and at 2 years by the same radiographer using a Lunar compared with a low salt diet (P 0.05 and 0.01, respectively) Prodigy DXA scanner (GE Medical Systems Inc., Madison, WI, USA). Daily o o phantom measurements were performed. Short-term precision was estimated and compared with 47 salt-resistant (SR) subjects on either salt 28 using duplicate measurements from seven postmenopausal women (from diet. Sharma et al. in cross-over studies showed that SS humans a separate study). CV was 0.5% for spine (L2–L4) and 0.6% for total hip. on a high salt diet also have significantly lower arterial blood pH (P 0.0001) and HCO (P 0.0001) levels than SR subjects. In our o 3 o Compliance measures. Compliance was estimated at each visit by capsule preliminary cross-over studies, significant increases in urinary Ca count for the potassium citrate and placebo groups, and by dietary excretion (corrected for creatinine excretion) associated with reporting and biomarkers (plasma vitamin C, whole-cell folate) for the diet increases in ADMA occurred in SS subjects on a high NaCl diet, but arm.38 not in SR subjects (R2 ¼ 0.47, P ¼ 0.02 vs R2 ¼ 0.03, P ¼ 0.57, 29 respectively) and not in the SS subjects on a low salt diet. Twenty-four-hour urine study. The women (n ¼ 70 at study start, n ¼ 57 at Recent studies have shown that giving NO mimetics may study end) collected 24-h urine samples at baseline, 3, 6, 12, 18 and 24 improve bone density. NO has been shown to prevent bone months for measurement of urinary pH and concentrations of potassium 30 breakdown in ovariectomized rats and women. A small study (K), Na, Ca, phosphate (PO4), urea and Cr. comparing isosorbide dinitrate 60 mg daily to alendronate 70 mg 31 weekly by Nabhan and Rabie demonstrated equivalent increases California in BMD in postmenopausal women after 1 year of treatment. More Subjects. Subjects were recruited from the San Francisco Bay area. The recently, an analysis of subjects from the Canadian Multicentre study was approved by the Institutional Review Board and all subjects Osteoporosis Study showed significant positive associations signed informed consent. Exclusion criteria were: renal or cardiac disease, 32 between nitrate users and higher BMD. parathyroid disease, previous bisphosphonate treatment, vertebral fracture Based on the evidence that sensitivity to salt as reflected by by history of an X-ray, BMDp À 2.5 on DEXA, o5 years past menopause, higher blood pressures for a given salt intake affects ADMA and hormone replacement therapy in the last 6 months or currently taking oral may differentially affect bone metabolism,29 we retrospectively corticosteroids or potassium-sparing diuretics. We included women taking tested correlations between blood pressure, NaCl and K alkali salt other types of diuretics or hypertension tablets and women on thyroxine intake and changes in bone markers or BMD in 266 treatment provided their thyroid function was within the normal range and medication doses unchanged in the last year. This study was started before postmenopausal (predominantly Caucasian) women who had clinical trial registration was required. Some of the data from this study has been subjects in osteoporosis studies in California and Scotland been published.39 and randomized to either placebo, potassium alkali salts or fruits and vegetables (latter Scotland only). Randomisation and intervention. The subjects were randomly assigned to one of four groups; one group received placebo pills and the other three arms were given active tablets containing 30, 60 or 90 mEq of potassium SUBJECTS AND METHODS bicarbonate (KHCO3). One-third of all subjects were randomized to placebo, one-third to 60 mmol of K and the remaining one-third to either North East (NE) Scotland 30 or 90 mmol of K. All subjects received Ca supplements (calcium Subjects. Subjects for this retrospective study were a subset of carbonate) if necessary to ensure a dietary intake of Ca of at least 1200 mg/ postmenopausal women living in the Aberdeen area who provided 24-h day (30 mmol/day) and a vitamin D supplement of 400 IU. We followed 11 urine samples during a dietary intervention trial. Exclusion criteria for the subjects at month 1 and then every 3 months for up to 36 months with original study, which was registered at http://www.controlled-trials.com as repeated blood samples and 24-h urine samples. ISRCTN86186352, were the lowest 10% of estimated dietary acidity (NEAP 33 calculated from the food frequency questionnaires), and any condition or Dietary intake and anthropometric assessments. Dietary intake of Ca and medication that would affect bone metabolism and potassium-sparing protein were estimated by questionnaires.40 Women were weighed (Scale- diuretics. Women taking other types of diuretics or hypertension tablets tronix, White Plains, NY, USA) and height measured with a stadiometer and women on thyroxine treatment provided their thyroid function was (Perspective Enterprises, Portage, MI, USA) at each visit associated with stable (as assessed by free T4 and TSH) and their dose had not changed in a DEXA study. Blood pressure was measured with an automatic the year before study entry were included in the study. Ethical permission sphygmomanometer (DinaMap, GE Healthcare/GE Medical Systems Inc., was obtained from Grampian Research Ethics Committee (02/0053). USA) and measured in the same arm at each visit after the patient was settled and before venipuncture was performed. Randomisation and Intervention. The subjects were randomized to one of the four groups: placebo, ‘low’ dose (18.5 mEq) or ‘high’ dose (55.5 mEq) Markers of bone health. Urine fDPD was measured in 2-h early morning potassium citrate, or fruit and vegetables (usual daily intake of fruit and fasted urine samples at baseline and every 3 months (Nichols Institute San vegetables plus 300 g). The women did not receive Ca or vitamin D Juan Capistrano, CA, USA). Cr was measured by colorimetric assay (COBAS, supplements. Roche) and the results are expressed as fDPD/Cr (nmol/mmol).

European Journal of Clinical Nutrition (2012) 1315 – 1322 & 2012 Macmillan Publishers Limited Blood pressure, salt and bone LA Frassetto et al 1317 BMD was measured at the spine and both hips at baseline and yearly Statistical analysis. All statistical analyses were carried out using SPSS using a Lunar Prodigy DXA scanner (GE Medical Systems Inc.). Daily quality version 17.0 (SPSS Inc., Chicago, IL, USA). Independent t-test for the assurance measurements were performed. continuous variables (with log transformation of the variable if required) and chi-squared test for categorical variables were used to test for Compliance measures. Compliance was judged by capsule counts every differences between the populations. Repeated measures analysis of 3 months and 24-h urinary K excretion. variance (ANOVA) were used to analyse changes in K excretion (as a measure of treatment compliance) and fDPD/Cr (bone resorption) over Twenty-four-hour urine study. Twenty-four-hour urine samples for K, Na, time. One-way ANOVA was used to analyse the 2-year change in BMD between treatment groups. Linear regression analysis was used to Ca, Cl, PO4 and Cr were obtained twice at baseline and then every 3 months. Samples for Na, K and Cl were measured by ion-selective determine predictors of blood pressure responses to salt that could be electrode and those for Ca, PO4 and Cr were measured by colorimetric potential confounders in the relationship between BMD change and salt assay at Quest Diagnostics (San Jose, CA, USA). intake. Analysis of covariance (ANCOVA) then allowed for the adjustment of these potential confounders (body mass index (BMI), 24-h urinary Ca excretion and 24-h urinary K excretion). Similarly ANOVA and ANCOVA All subjects were used to explore the cross-sectional relationship between tertiles of Blood pressure determinations. For each individual, the mean arterial MAP/Na and baseline BMD. pressure (MAP) was calculated from the equation: MAP ¼ diastolic blood When the data from both centres were combined, the 18.5 mmol dose pressure þ 1/3(systolic À diastolic blood pressures). The women were from NE Scotland was combined with the 30 mmol dose of the California divided into tertiles of low, medium or high MAP at the baseline visit. centre to give a low K salt group, and the 55.5 mmol dose from NE MAP was also divided by 24-h urinary Na excretion as a measure of blood Scotland was combined with the 60 mmol dose from California to give a pressure response to salt intake; MAP/Na. Change in MAP was calculated as high K salt group. Both centres contributed to the combined placebo the average of all MAPs on treatment minus the average of all MAPs before group. treatment.

PRAL and NAE calculations. For the Aberdeen cohort, we estimated urine RESULTS PRAL from the formula: A total of 266 postmenopausal women attended the baseline visit. ½ðÞþdiet proteinÂ0:48 measured urine PO4 Compared with the Scottish women (n ¼ 70), the Californian À ½measured urine K þ measured urine Ca þ ðÞ0:026Âdiet Mg : women (n ¼ 196) were marginally older, taller; had lower BMI but they had similar biochemical profiles (Table 1). Overall, the Diet net acid load was estimated as PRAL þ OA production, where PRAL experimental intervention was completed by 209 women was calculated from the estimated dietary intakes and OA production (78.5%) over 2 years and the withdrawal rate was similar across using the body surface area method: the treatment groups (Figure 1). Estimated NEAPðÞ¼ mEq/d PRALðÞþ mEq/d OAestðÞ mEq/d 41 Figure 2 gives an estimate of treatment compliance for both centres, as measured by 24-h urinary K excretion. Overall Also for that group, we estimated diet net acid load from the formula using compliance was good with significant increases in K excretion 33 estimates of dietary protein and potassium: from baseline according to the dose of K salt (repeated measures 62:1ÂðÞÀdiet Pro/diet K 17:9 ANOVA Po0.001) for the combined centres (California Po0.001, NE Scotland P ¼ 0.004), although there was a temporary decrease For the California cohort, we estimated urine PRAL from the formula: at 6 months for the 90-mEq group. MAP in mm Hg at baseline for the combined centres were ½ðÞþdiet proteinÂ0:48 measured urine PO4 82.3±5.2 (low), 94.3±3.3 (medium) and 110.3±8.5 (high) (one- measured urine K measured urine Ca measured urine Mg : À ½þ þ way ANOVA Po0.0001). Of the 266, 20 (8%) were on blood Diet net acid load was estimated using the Frassetto formula above, pressure medications. A sensitivity analysis with and without this modified so that we used urine K as an estimate of diet K. We had group was unchanged. For the Californian women, the numbers of insufficient dietary data to calculate dietary PRAL. women defined as having low, medium and high blood pressures

Table 1. Baseline characteristics of subjects providing 24-h urine samples

Characteristics NE Scotland mean±s.d. (min, max) California mean±s.d. (min, max) P-valuea

Number of subjects 70 196 Age (years) 59.7±2.2 (55.3, 65.4) 62.2±6.8 (46.3, 80.1) o0.001 Weight (kg) 74.5±1.3 (48.5, 105.0) 70.0±1.1 (41.1–96.2) 0.104 Body mass index (kg/m2) 30.0±5.0 (21.2, 44.5) 24.9±3.9 (17.5, 37.0) 0.002 Height (cm) 160.4±5.4 (148.4, 172.8) 163.6±6.6 (147.5, 180.0) 0.032 Systolic blood pressure (mm Hg) 131.5±17.3 (102, 197) 134.1±22.3 (91, 201) 0.008 Diastolic blood pressure (mm Hg) 80.1±12.4 (60, 127) 75.3±10.6 (50, 109) 0.211 MAP (mm Hg) 97.2±12.8 (76, 150) 94.9±13.3 (68, 133) 0.345 24-h urinary creatinine (mmol/l) 8.3±2.3 (3.3, 13.6) 9.9±2.4 (0.4, 20.7) 0.753 24-h urinary sodium (mmol) 123.4±51.6 (28, 337.5) 116.4±41.2 (23.3, 267.3) 0.107 Serum creatinine (mmol/l) 84.9±9.4 (67.0, 118.0) 70.7±9.8 (42.4, 102.5) 0.401 Calculated creatinine clearance (ml/min) 74.2±15.1 (46.6, 125.8) 77.1±18.0 (43.3, 123.1) 0.052 Lumbar spine (L2–L4) BMD (g/cm2) 1.182±0.186 (0.94, 1.82) 0.906± 0.135 (0.62, 1.41) 0.923 Left hip BMD (g/cm2) 0.982± 0.118 (0.76, 1.39) 0.802±0.112 (0.49, 1.18) 0.162 Urinary fDPD/Cr (nmol/mmol) 5.99±2.23 (2.89, 19.63) 5.49±2.71 (0.55, 31.41) 0.006 Ethnicity (% Caucasian, Asian, Hispanic) 100, 0, 0 88, 9, 3 0.009 Blood pressure-lowering medication (n, %) 18 (26%) 4 (2%) 0.002 Abbreviations: BMD, bone mineral density; Cr, creatinine; fDPD, free deoxypyridinoline; MAP, mean arterial pressure; NE Scotland, North East Scotland. aIndependent t-test for the continuous variables (with log transformation of the variable if required) and chi-squared test for categorical variables.

& 2012 Macmillan Publishers Limited European Journal of Clinical Nutrition (2012) 1315 – 1322 Blood pressure, salt and bone LA Frassetto et al 1318 North East Scotland

Randomized n=70

K Citrate 55.5meq/d K Citrate 18.5meq/d Placebo Fruit and Veg n=18 n=16 n=17 n=19 n=18 n=16

Completed study visits n=65

n=16 n=14 n=17 n=18

California

Randomized n=196

KBC 60 mmol/d KBC 90 mmol/d KBC 30 mmol/d Placebo n=63 n=30 n=35 n=68 n=16

Completed study visits n =144

n=24 n=47 n=25 n=48

Figure 1. Details of study recruitment and randomisation for the NE Scotland and California. K citrate, potassium citrate; KHCO3, potassium bicarbonate.

in relation to salt intake were 66, 65 and 65, respectively. The (n ¼ 49), 55.5 mmol and 60 mmol to the ‘high-dose K salt’ treatment corresponding numbers for the Scottish women were 22, 25 and (n ¼ 76) and a combined placebo group (n ¼ 95)) did not change 23 (Table 2). At baseline, the subjects who had the lowest blood the outcome (ANCOVA adjusting for urinary K and Ca excretion and pressure in relation to Na intake had the highest urinary Na BMI. For hip: P ¼ 0.119 for low MAP/Na, P ¼ 0.203 for medium MAP/ excretion (one-way ANOVA Po0.0001) and the highest urinary K Na and P ¼ 0.997 for high MAP/Na groups. For LS, P ¼ 0.959 for low and Ca excretion (Po0.01, Po0.02, respectively). Linear regression MAP/Na, P ¼ 0.117 for medium MAP/Na and P ¼ 0.923 for high analysis of all subjects showed that 24-h urinary K and Ca MAP/Na groups). excretion, and BMI were significant predictors of MAP/Na For all women, regardless of the blood pressures responses to accounting for 8.5%, 4.2% and 1.3% of the total variation, Na, 3-monthly urinary measures of bone resorption did not differ respectively (full data not shown). These variables were then according to dose of K salt (repeated measures ANOVA: NE included as confounders in the following ANCOVA analysis. The Scotland P ¼ 0.858, California P ¼ 0.762) (Table 3). When testing California group also had 24-h urine chloride measurements the cross-sectional relationship between MAP/Na and bone evaluated; there was no association with MAP (P ¼ 0.08). There health: for NE Scotland baseline, BMD was observed to be higher was a significant inverse correlation between change in MAP and in the lowest category of MAP/Na compared with the other K intake with treatment (r ¼À0.20, Po0.05). categories, but this was not statistically significant (P ¼ 0.061); for The overall percentage bone loss±s.e.m. for NE Scotland was the California centre, there was no relationship (P ¼ 0.165); and À 1.5±2.4% for the hip, À 2.6±3.5% for LS (lumbar spine); and for adjustments for confounders did not change the outcome for California it was À 0.6±2.8% for the hip and 0.5%±3.4% for LS. either centre (Table 2). Further, if the two centres were combined, The differences between centres was significant (P ¼ 0.027 hip, there was no statistically significant association with or without Po0.001 spine). We found no difference in 2-year percentage adjustments for confounders, including study centre (data not change in BMD, either at the hip or spine in relation to K salt shown). treatment, for either centre. The response did not differ according Data for the dietary estimates of net acid production and to blood pressure in relation to sodium intake (Figures 3a–d). excretion at baseline are shown in Table 4. The estimates between Adjustment for confounders did not change the relationship. the two cohorts for the diet net acid production and urinary PRAL Further, combining the data from both centres to maximise power are quite similar, and similar to other estimates of dietary net acid (with 18.5 mmol and 30 mmol contributing to ‘low-dose K salt’ excretion for Western diets.6,33,41

European Journal of Clinical Nutrition (2012) 1315 – 1322 & 2012 Macmillan Publishers Limited Blood pressure, salt and bone LA Frassetto et al 1319 46 North east scotland and bone strength. These ﬁndings support the latter 160 investigators’ earlier study that reported treatment with K citrate Placebo attenuated 1-year bone loss compared with K chloride.43 A 140 Fruit and vegetable number of meta-analyses have questioned the hypothesis that the 10,47,48,49 18.5 diet net acid load reduces bone mass. Mean estimated 120 dietary NEAP is in the order of þ 40 mEq/day, whereas estimated 55.5 urinary PRAL is low. If these women were eating generally healthy 100 diets, although dietary acidity was positive, it was perhaps 80 insufﬁciently acidic enough (as indicated by the negative urinary PRAL) so that the additional alkaline-generating potassium salts

60 did not beneﬁt BMD. 24 hr potassium hr 24

excretion (mmol) excretion Although our data fail to show that moderately high alkali diets 40 are beneﬁcial to bone health in younger healthy postmenopausal Caucasian women, other subgroups of subjects might respond to 20 alkali therapy, for example, older subjects with more age-related renal dysfunction41 or those with metabolic syndrome. Presence 0 of risk factors for the metabolic syndrome is associated with both Baseline 3 6 12 18 24 higher blood pressures with higher salt intake50 and higher Timepoint fracture risk.51 Metabolic syndrome has been associated with decreased production of NO, and NaCl has been shown to dose California dependently decrease endothelial NO synthase activity in 52 160 endothelial cells. NO synthases have been demonstrated in both osteoclasts and osteoblasts.53,54 NO may be the mediator by 140 which oestrogen or androgen therapy improves BMD. Nitrate use is associated higher BMD32 and prevents bone loss in 30,31 120 postmenopausal women. The subjects in our study had relatively few risk factors for metabolic syndrome. Although the 100 women in the Scottish cohort tended to be heavier than the California cohort, and we do not have information on lipid proﬁle, 80 only a small percentage of the women were taking medications for blood pressure or had abnormal fasting glucose measures.

60 Another explanation for the null finding is that the effect size of 24 hr potassium hr 24 excretion (mmol) excretion Placebo dietary acid and salt is small compared with the effect size of other 40 factors that affect bone metabolism. In the Study of Osteoporotic 30 Fractures,42 the observational analyses had to be corrected for 60 20 age, energy intake, total Ca intake (dietary Ca plus supplements), 90 total protein intake, weight, current oestrogen use, physical 0 activity, smoking status and alcohol intake before associations Baseline 3 9 15 21 27 33 with potential acid intake could be detected. The subjects in the Study of Osteoporotic Fractures were a decade older than our Timepoint subjects and had age-related decline in renal function. In contrast, Figure 2. Twenty-four-hour urinary K excretion for the two centres. the women in our intervention studies were apparently healthy. In population studies, higher K intake is associated with lower blood pressures.22,55 Among our subjects, those whose blood pressures were the lowest at baseline, independent of Na intake had the highest urinary K excretion, and increasing K intake with DISCUSSION treatment lowered blood pressure. Meta-analyses of calcium Contrary to our hypothesis that blood pressure responses to NaCl supplementation trials have also shown a slight lowering of intake (as measured by MAP and MAP/24-h urinary Na excretion) blood pressures,56 but adjusting for baseline calcium excretion did could help predict bone response to alkali therapy, in this not change our results. population of predominantly healthy Caucasian postmenopausal There were some limitations of our study. First, there is no women, no differences in BMD at the hip or spine or differences in agreed upon definition of what constitutes a blood pressure urinary fDPD excretion were seen between treatment groups. The response to salt.57 In typical salt-sensitivity metabolic studies, women were enrolled for at least 2 years in studies designed to subjects’ blood pressures are measured on a low salt diet and then investigate the effects of dietary alkali intake on bone health. The on a high salt diet, with sensitivity to salt being defined by how bone loss observed for NE Scotland for the 24-h urine subset was much the subjects blood pressure increased, usually 3–5 mm Hg. similar to that already reported for the full data set.11 This is the As this was a retrospective analysis; this was not possible. first time the Californian data relating to bone health have been However, average MAP differences between our three tertiles shown. The percentage bone loss was less compared with NE were 12–16 mm Hg and significantly different from each other. Scotland, which may be because the Californian women were Second, we were unable to analyse some bone markers, such as more than 5 years past the menopause. C-telopeptide (CTX) or propeptide type 1 procollagen (P1NP), Earlier studies evaluating the potential effects of high alkali which were not done in the California study; recent literature diets on BMD and fracture risk have had conflicting results. Some suggests that CTX and P1NP are more precise bone markers than observational studies suggest a role for acid–base balance on urine fDPD.58 Although the Scottish study followed 260 women, fractures42 and others do not.10 Intervention studies of 1 year or only 70 of them had 24-h urine studies collected, and urine less suggest a positive effect43,44,45 on markers of bone turnover. chloride was not done. Third, the California women received a Whereas one 2-year placebo-controlled trial did not find an effect calcium and vitamin D supplement, whereas the Scottish women of K citrate on BMD,11 a more recent study found increases in BMD did not. However, analyses of the two studies separately did not

& 2012 Macmillan Publishers Limited European Journal of Clinical Nutrition (2012) 1315 – 1322 Blood pressure, salt and bone LA Frassetto et al 1320 North East Scotland California

Dose 0 Fruit 18.5 55.5 Dose 0 30.0 60.0 90.0 and veg 4 4 2 2 0 0 -2 -2 -4 -4 -6 -6 % change in hip BMD % change in hip BMD -8 -8

North East Scotland California Dose 0 Fruit 18.5 55.5 Dose 0.0 30.0 60.0 90.0 and veg 6 6 4 4 2 2 0 0 -2 -2 -4 -4 -6 Low MAP/Na -6 Medium MAP/Na -8 High MAP/Na % change L2 to L4 BMD

% change L2 to L4 BMD -8 -10 -10 Figure 3. (a) Mean % change in left hip BMD over 24 months (±s.e.m.) for the NE Scottish subjects with K alkali treatment according to tertile of MAP/Na (n ¼ 22, 25, 23 for low, medium and high, respectively, ((±s.d.), low MAP/Na ¼ 0.55±0.097, medium MAP/Na ¼ 0.81±0.078 and high MAP/Na ¼ 1.45 ±0.50 mm Hg/mmol Na) and P ¼ 0.167 for low MAP/Na, P ¼ 0.803 for medium MAP/Na and P ¼ 0.743 for high MAP/Na groups (one-way ANOVA)). After adjustment for urinary potassium, calcium and BMI, P ¼ 0.299, P ¼ 0.748, and P ¼ 0.715, respectively for low, medium and high SS groups (ANCOVA). Overall mean % change in hip BMD À 1.7± 3.2% low MAP/Na, À 1.4±2.2% medium MAP/Na and À 1.4±1.5% high MAP/Na. (b) Mean (±s.e.m.) % change in left hip BMD over 24 months for the Californian subjects with K alkali treatment according to tertile of salt sensitivity (n ¼ 66, 65, 65 for low, medium and high MAP/Na, respectively, ((±s.d.), low MAP/Na ¼ 0.58±0.097, medium MAP/Na ¼ 0.85±0.083 and high MAP/Na ¼ 1.40±0.51 mm Hg/mmol Na) and P ¼ 0.423 for low MAP/Na, P ¼ 0.078 for medium MAP/ Na and P ¼ 0.887 for high MAP/Na groups for mean change in hip BMD (one-way ANOVA)). After adjustment for urinary potassium, calcium and BMI, P ¼ 0.385, P ¼ 0.147 and P ¼ 0.971, respectively, for low, medium and high MAP/Na groups (ANCOVA). Overall mean % hip BMD À 0.2±2.4% low MAP/Na, À 1.4±3.0% medium MAP/Na and À 0.5±2.7% high MAP/Na. (c) Mean % change in lumbar spine BMD over 24 months (±s.e.m.) for the NE Scottish subjects (MAP/Na details as for panel (a)) and P ¼ 0.113 for low MAP/Na, P ¼ 0.730 for medium MAP/Na and P ¼ 0.586 for high MAP/Na groups (one-way ANOVA). After adjustment for urinary potassium, calcium and BMI, P ¼ 0.171, P ¼ 0.849 and P ¼ 0.840, respectively, for low, medium and high MAP/Na groups (ANCOVA). Overall mean % LS BMD À 3.3±2.9% low MAP/Na, À 1.5±3.4% medium MAP/Na and À 3.3±4.1% high MAP/Na. (d) Mean (±s.e.m.) % change in lumbar spine BMD over 24 months for the Californian subjects (MAP/Na details as for panel (b)) and P ¼ 0.311 for low MAP/Na, P ¼ 0.152 for medium MAP/Na and P ¼ 0.870 for high MAP/Na groups for mean change in lumbar spine BMD (one-way ANOVA). After adjustment for urinary potassium, calcium and BMI, P ¼ 0.318, P ¼ 0.363 and P ¼ 0.883, respectively, for low, medium and high MAP/Na groups (ANCOVA). Overall mean % LS BMD À 0.8±2.7% low MAP/Na, À 0.3±3.3% medium MAP/Na and À 0.5±4.7% high MAP/Na.

Table 2. Urinary salt excretion, blood pressure and BMI according to tertile of MAP/Na

Mean±s.d. Tertile of MAP/UNa P-value

LS MS HS

North East Scotland n 22 25 23 MAP/UNa (mm Hg/mmol Na) 0.55±0.097 0.81±0.078 1.45±0.50 MAP (mm Hg) 96.0±11.7 96.6±11.2 98.7±15.7 0.771 Diastolic blood Pressure (mm Hg) 79±12 81±11 81±15 0.841 Systolic blood Pressure (mm Hg) 131±15 130±17 135±19 0.573 Na (mmol/24 h) 180±44 120±16 73±19 o0.001 K (mmol/24 h) 74±21 62±21 46±17 o0.001 Ca (mmol/24 h) 4.1±2.2 3.4±1.5 2.4±1.2 0.004 BMI (kg/m2) 29.7±5.2 29.4±6.0 27.8±3.5 0.399

California n 66 65 65 MAP/UNa (mm Hg/mmol Na) 0.58±0.097 0.85±0.083 1.40±0.51 MAP (mm Hg) 90.6±13.1 95.5±12.2 98.7±13.5 0.002 Diastolic blood Pressure (mm Hg) 72±11 76±10 78±10 0.003 Systolic blood Pressure (mm Hg) 128±21 133±20 140±25 0.006 Na (mmol/24 h) 159±30 112±16 76±21 o0.001 K (mmol/24 h) 75±23 66±21 62±27 0.009 Ca (mmol/24 h) 4.3±2.3 3.9±1.8 3.2±1.8 0.014 BMI (kg/m2) 25.9±3.7 24.6±4.1 24.2±3.7 0.032 Abbreviations: BMI, body mass index; HS, high salt; LS, low salt; MAP, mean arterial pressure; MS, moderate salt; UNa, urinary Na. MAP calculated from MAP ¼ diastolic blood pressure þ 1/3(systolic À diastolic blood pressures).

European Journal of Clinical Nutrition (2012) 1315 – 1322 & 2012 Macmillan Publishers Limited Blood pressure, salt and bone LA Frassetto et al 1321 Table 3. Mean (±s.d.) fDPD/Cr measurements in nmol/mmol for all subjects split by dose and visit.

Time Potassium salt dose, mEq/day (North East Scotland)a Potassium salt dose, mEq/day (California)a

0 (Placebo) Fruit and 18.5 55.5 Pb 0 (Placebo) 30 60 90 Pb vegetable (ca. 18.5)

n 16 18 15 16 67 35 63 30 Baseline 5.68±1.8 5.67±1.45 5.63±1.17 6.98±3.58 0.24 5.12±2.02 5.55±2.12 5.79±3.90 5.59±1.22 0.56 n 15 10 11 15 64 34 58 29 1 month 5.27±1.41 5.37±2.13 5.42±0.86 5.24±1.80 0.99 5.58±4.56 5.36±2.51 4.91±2.08 5.74±2.34 0.62 n 17 19 14 18 63 33 56 26 3 months 5.55±1.66 6.16±2.02 5.53±1.01 5.33±1.11 0.40 5.26±2.18 5.33±1.86 5.13±2.47 5.76±1.77 0.66 n 14 18 12 16 59 31 52 27 6 months 5.09±1.54 6.12±1.88 5.52±1.01 5.96±1.48 0.26 5.53±2.18 5.02±1.69 5.11±1.68 5.17±2.06 0.57 n 16 19 14 14 60 30 53 25 12 months 5.53±1.49 5.51±1.54 5.65±1.51 5.21±1.12 0.58 5.08±1.91 4.71±1.62 5.11±1.94 5.42±1.77 0.26 n 13 15 12 15 54 26 49 26 18 months 6.28±1.94 5.60±1.64 5.45±1.41 5.13±1.01 0.27 4.81±2.13 4.16±1.12 4.28±1.23 4.32±1.29 0.24 n 17 18 14 16 48 25 47 24 24 months 5.50±1.89 5.51±1.05 5.10±0.80 5.95±2.30 0.58 4.21±1.63 4.17±1.36 4.15±1.31 4.45±2.04 0.89 Abbreviations: Cr, creatinine; fDPD, free deoxypyridinoline. aPotassium salt was potassium citrate for NE Scotland and potassium bicarbonate for California; bOne-way ANOVA

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European Journal of Clinical Nutrition (2012) 1315 – 1322 & 2012 Macmillan Publishers Limited