Bone 44 (2009) 120124

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Bone
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b o n e

Alkaline mineral water lowers bone resorption even in calcium sufciency:

Alkaline mineral water and bone metabolism
Emma Wynn a,, Marc-Antoine Krieg a, Jean-Marc Aeschlimann b, Peter Burckhardt c
a
Centre of Bone Diseases, Lausanne University Hospital, Switzerland
b
Nestl Research Centre, Switzerland
c
Clinic Bois-Cerf, Osteoporosis Consultation, Lausanne, Switzerland

a r t i c l e i n f o a b s t r a c t

Article history: Background: Dietary acid charge enhances bone loss. Bicarbonate or alkali diet decreases bone resorption in
Received 14 January 2008 humans. We compared the effect of an alkaline mineral water, rich in bicarbonate, with that of an acid one,
Revised 22 August 2008 rich in calcium only, on bone markers, in young women with a normal calcium intake.
Accepted 10 September 2008
Methods: This study compared water A (per litre: 520 mg Ca, 291 mg HCO3, 1160 mg SO4, Potential Renal Acid
Available online 26 September 2008
load (PRAL) +9.2 mEq) with water B (per litre: 547 mg Ca, 2172 mg HCO3, 9 mg SO4, PRAL 11.2 mEq). 30
Edited by: R. Baron female dieticians aged 26.3 yrs (SD 7.3) were randomized into two groups, followed an identical weighed,
balanced diet (965 mg Ca) and drank 1.5 l/d of the assigned water. Changes in blood and urine electrolytes, C-
Keywords: telopeptides (CTX), urinary pH and bicarbonate, and serum PTH were measured after 2 and 4 weeks.
Bicarbonate Results: The two groups were not different at baseline, and showed a similar increase in urinary calcium
Mineral water excretion. Urinary pH and bicarbonate excretion increased with water B, but not with water A. PTH (p = 0.022)
Nutrition and S-CTX (p = 0.023) decreased with water B but not with water A.
PRAL Conclusion: In calcium sufciency, the acid calcium-rich water had no effect on bone resorption, while the
PTH
alkaline water rich in bicarbonate led to a signicant decrease of PTH and of S-CTX.
Resorption markers
2008 Elsevier Inc. All rights reserved.

Introduction In vitro, an efux of calcium from bone tissue is already noted after
3 h incubation in an acidic medium [15]. Indeed, bone has been shown
The benecial effects of numerous mineral waters on bone to contribute to the maintenance of the extra-cellular uid pH [16]. A
metabolism are mainly attributed to their calcium content [16]. low acid diet could contribute to the maintenance of bone mineral
Calcium-rich mineral waters are an alternative to dairy products as density [17]. In healthy subjects, supplements of potassium bicarbo-
their bioavailability is similar or even better [7]. They have decreased nate, potassium citrate, and even mineral water rich in bicarbonates
bone resorption, but only in short-term trials or in calcium and decreased the calciuria and bone resorption markers [1821].
oestrogen deciency [3]. Minerals other than calcium, especially We investigated whether a high bicarbonate and calcium intake
bicarbonate, also have a positive effect on bone metabolism [8,9]. from an alkaline mineral water decreases bone resorption more than a
Nutritional acid load contributes to the age related bone loss [10]. To high calcium intake from an acid water, in healthy subjects on a
keep pH constant, the organism uses buffer systems such as bone, balanced diet adapted to their energetic needs.
expiration of CO2 and renal excretion of acids [11]. The endogenous
production of acid, consequent to a normal Western diet, is about 1 Subjects and methods
mEq/kg body weight, per day [12]. To buffer this, 2 mEq of calcium is
necessary [12]. The more acid precursors a diet contains, the greater For this open, randomized, controlled study, thirty female
the need for buffering [13] and the enhancement of bone resorption. dieticians or student dieticians were recruited. The inclusion criteria
In addition, with advanced age, the ability to excrete acids by the were good health, 1845 years and BMI 18.525 kg/m2. The exclusion
kidney declines [14]. criteria were medication interfering with calcium metabolism
(diuretics, corticoids), pregnancy, and inclusion in other protocols.
They were declared healthy after completion of a medical ques-
This study was funded by the Foundation for Research on Osteoporosis and Bone tionnaire. Out of the 30 participants, 23 were on the contraceptive pill.
Diseases, Lausanne, Switzerland. The manufacturers generously provided the mineral The subjects were randomized into 2 groups using the method of
water.
* Corresponding author. DAL-Hpital orthopdique, Centre des Maladies Osseuses,
randomly permuted blocks and assigned to mineral water A or B. 4 of
Avenue Pierre-Decker 4, 1011 Lausanne, Switzerland. Fax: +41 021 314 47 37. the 7 subjects not taking a contraceptive pill were randomly attributed
E-mail address: emma.wynn@chuv.ch (E. Wynn). to Water A and 3 to Water B.

8756-3282/$ see front matter 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.bone.2008.09.007
 E. Wynn et al. / Bone 44 (2009) 120124 121

Each subject had to drink 1.5 l a day of one of the two mineral day, not to drink alcohol and to drink a little tap water if they were
waters during 28 days. really too thirsty. A glass of fruit juice had to be considered as a portion
The composition of the waters is shown in Table 1. One is very rich of fruit, and a glass of milk as a portion of dairy product. Subjects were
in calcium (Water A, AdelbodnerR, Switzerland) and the other is asked to avoid salty processed food and to moderately salt their food.
equally rich in calcium but also rich in bicarbonate (Water B, The three following visits occurred early morning of the rst day, at
KryniczankaR, Poland). In addition, water A was also rich in sulphate 2 weeks and at 4 weeks of the study. They enabled reception of the 24
which increases the acid load. For the assessment of the acid or h urine, collection of the fasting morning urine and blood sampling.
alkaline load of food, a calculation model was developed by Remer and
Manz [22], the potential renal acid load (PRAL). PRAL can be accurately Blood tests
calculated when the nutrient data for protein, P, Cl, K, Mg, Ca and Na is
known. Phosphate content of mineral water is negligible and for this The following analyses were undertaken at baseline and at 2 and 4
reason not indicated. On request, both manufacturers conrmed that weeks: sodium, potassium, ionized and total calcium, phosphorus,
the phosphate content of their waters is non signicant. Therefore, alkaline phosphatase, albumin, creatinine, 25-OH vitamin D (DiaSorin,
phosphate was not included in the PRAL algorithm. PRAL takes into Stillwater, Minnesota, 55082-0285 USA, (RIA+ extraction), intra- and
account the average intestinal absorption rate of the respective com- interassay coefcients 8.612.5k and 6.113.2k, detection limit 2 g/l),
ponents. Its algorithm is (0.00049 protein[mg]) + (0.027 Cl [mg]) + blood and urinary C-telopeptides (CTX), PTH (Intact PTH, Nichols
(0.037 P [mg]) (0.021 K [mg]) (0.026 Mg [mg]) (0.0413 Na [mg]) Institute, San Clemente, CA 92673, USA, (CIA), intra- and interassay
(0.013 Ca [mg]) [22]. This algorithm takes into account methionine coefcients 5.76.2k at and 7.39.7k, detection limit 2 ng/l), glucose
and cysteine as nutritional sources of SO4 coming from protein. (baseline only) and blood cell count (baseline only). The electrolytes,
However in mineral waters, SO4 is in solution. This justies a albumin and alkaline phosphatase were analysed at the routine
correction of the formula by using the molecular weight of SO4 Laboratory of the hospital using standard methods. CTX (C-terminal
instead of the two amino acids which is 96, and an absorption rate of fragment of the type I collagen) were assayed with the Beta-Cross Laps
70k, resulting in a conversion factor of 0.0146 instead of 0.00049 Elecsys, Roche Diagnostics GmbH, D-68293 Mannheim, (ECLIA), intra-
(Remer T, personal communication, 2007). Therefore, the PRAL is +9.2 and interassay coefcients 1.64.7k and 3.94.3k, detection limit 0.010
mEq/l for water A (acid) and 11.2 mEq/l for water B (alkaline). g/l) at the Endocrinology Laboratory of the hospital. All the samples were
The subjects were informed of the aims of the study and gave hermetically closed at reception and transported to the lab in less than
written consent. 15 min. The measurement of ionised calcium was introduced during the
Our protocol was accepted by the Ethic Committee of the Faculty of trial. For this reason it was performed only in 5 subjects on water A and 7
Medicine of the University of Lausanne, Switzerland. The study took subjects on water B. A multiple specic electrode automate was used
place at the University Hospital. (ABL 800, Radiometer, Copenhagen, RSCH).
Prior to the rst of the four visits at the hospital, the subjects were
given full explanations by phone about the study and were questioned Urinary tests
on their health status to identify inclusion and exclusion criteria. 35
volunteers applied and 30 could be included. Five were not included Calcium, sodium, potassium, creatinine, bicarbonate and pH were
as they lived too far away to be on time for the early morning measured in the 24 h urine and in the 2 h fasting morning urine
appointments. At the rst visit the subjects got the bottles of mineral (second miction). In addition, CTX were measured (Nordic Bioscience
water and they met a qualied dietician (EW) who explained the Diagnostics A/S, Herlev-Hovedgade 207, 2730 Herlev, Denmark,
imperatives of the study, i.e. mainly the dietary recommendations and (ELISA), intra- and interassay coefcients 4.79.4k and 6.616.3k,
delivered the same weighed and balanced diet plan for 28 days to all detection limit 0.050 mg/l). Urine gas analysis was done with a
subjects. The diet plan followed the recommendations of the daily gazometer (Bayer Diagnostics 248) at the Paediatric Research
consumption frequencies of 5 portions of fruit and vegetables, 3 of Laboratory.
starch, 3 of dairy products and 1 of meat, sh or egg. The diet plan
included 1875 kcal, 237 g of carbohydrate, 67 g of lipids, 75 g of protein Statistical analysis
and 965 mg of calcium. Subjects were allowed to eat more or less
starch depending on their appetite and physical activity. The other The results were analysed using ExcelR functions, StatistiX 8
food groups had to be consumed in the established quantities. All (Analytical Software, Tallahassee, FL, USA) and SPSS version 14.0 (SPSS
participants had to drink 1.5 l a day of the assigned mineral water. Inc., Chicago, IL, USA). Differences were considered signicant at
They were advised not to drink more than one cup of coffee or tea a p b 0.05. Descriptive statistics were undertaken for all anthropometric
parameters and analytical variables. For each variable, groups'
homogeneity was checked at baseline by Student t-tests. Groups
were compared using paired changes from baseline by classical t-tests
Table 1
Composition of the mineral waters (Mean and SD), robust ones (Median and robust SD), as well as by
Wilcoxon rank-sum tests. Results from these 3 tests being comparable,
Water A Water B
we only present the classical t-tests (Mean and SD).
mg/l mmol/l mEq/l mg/l mmol/l mEq/l
Calcium 520 13 26 548 13.7 27.4 Results
Magnesium 35 1.4 2.9 65 2.7 5.4
Sodium 5.5 0.2 0.2 67 2.91 2.91
Potassium 1.55 0.04 0.04 6.5 0.2 0.2 The two groups were comparable in age (group A: 26.3 yrs 7.6;
Chloride 5.6 0.2 0.2 10.6 0.3 0.3 group B: 26.3 yrs 7.3) and BMI (group A: 20.6 1.8 kg/m2; group B:
Sulphate 1160 12.1 24.2 9 0.09 0.18 21.3 2.3 kg/m2). The blood formula and the glycaemia were all in the
PRALa +9.2 11.2
reference limits. There were no signicant differences among the two
Fluoride 0.2 0.011 0.011 0.1 0.005 0.005
Bicarbonate 291 4.8 4.8 2172 35.6 35.6 groups in any of the laboratory values, as well as in age, height, weight
and BMI.
Manufacturers always indicate the composition of mineral waters in mg/l.
Results of this trial are indicated in mmol/l for comparison with the literature.
Being dieticians, the participants showed excellent compliance and
PRAL in mEq/l allows to assess the acid or the alkaline load. perfect understanding of the diet plan. All 30 volunteers completed
a
PRAL according to Remer and Manz ([22], Remer T, personal communication, 2007). the study.
122 E. Wynn et al. / Bone 44 (2009) 120124

Table 2
Biochemical results (mean SD)

Units Baseline value (n = 15) 2 weeks (n = 15) 4 weeks (n = 15)

Serum
PTH Water A ng/l 28.6 (11.6) 27.0 (9.4) 29.1 (15.9)
Water B 33.3 (11) 26.1 (10) 27.7 (8.1)
CTX Water A g/l 0.380 (0.115) 0.385 (0.137) 0.389 (0.110)
Water B 0.479 (0.261) 0.413 (0.198) 0.401 (0.234)
Vitamin D Water A g/l 23.1 (6.3) 22.4 (7.7) 23.6 (7.3)
Water B 25.5 (10) 24.5 (10.3) 25.8 (9.6)
Phosphatase alkaline Water A U/l 53.0 (14.2) 53.8 (16.8) 51.3 (13.9)
Water B 49.1 (11.8) 49.2 (11.2) 46.7 (11.8)
Phosphorus Water A mmol/l 1.26 (0.16) 1.28 (0.16) 1.27 (0.12)
Water B 1.28 (0.18) 1.31 (0.2) 1.29 (0.14)

24 h urinary excretion
pH Water A 6.3 (0.46) 5.9 (0.34)* 6.0 (0.35)
Water B 6.0 (0.50) 6.7 (0.29) 6.6 (0.43)#
Bicarbonate Water A mmol/24 h 4.9 (5.52) 1.3 (0.72)* 2.1 (1.92)
Water B 2.7 (2.36) 13.0 (7.84) 13.8 (6.67)#
Ca Water A mmol/24 h 4.7 (1.80) 5.6 (1.48) 5.5 (1.16)
Water B 3.8 (1.90) 4.5 (1.41)* 4.5 (1.58)#
Na Water A mmol/24 h 123 (35) 110 (30) 119 (41)
Water B 120 (25) 143 (57) 146 (47)
K Water A mmol/24 h 69.4 (26) 67.4 (8.6) 73.4 (18.5)
Water B 60.1 (18.1) 70.5 (20.1) 78.7 (21.3)*
Creatinine Water A mol/24 h 9779 (2362) 9975 (1639) 10515 (1635)
Water B 9619 (1781) 10143 (1415) 10542 (1823)
CTX Water A mg/24 h 2.8 (1.02) 2.8 (1.42) 2.7 (0.71)
Water B 3.1 (2.04) 3.2 (1.41) 3.1 (1.46)

2 h fasting urine
pH Water A 5.7 (0.52) 5.6 (0.36) 5.5 (0.47)
Water B 5.6 (0.5) 6.0 (0.7) 6.3 (0.6)#
Bic/Creat. Water A mmol/mmol 0.14 (0.20) 0.08 (0.06) 0.08 (0.09)
Water B 0.11 (0.13) 0.32 (0.45) 0.75 (1.09)*#
Calcium/Creat. Water A mmol/mmol 0.25 (0.15) 0.29 (0.16) 0.22 (0.13)
Water B 0.21 (0.13) 0.20 (0.11) 0.17 (0.09)
CTX/Creat. Water A mg/mmol 0.066 (0.040) 0.060 (0.041) 0.048 (0.026)
Water B 0.086 (0.066) 0.060 (0.039) 0.079 (0.098)

Differences from baseline: *p b 0.05, p b 0.01 p b 0.001.

Differences between Water A and B: #p b 0.05.

Laboratory tests groups: phosphorus, potassium, sodium, calcium, ionised calcium,

alkaline phosphatase, albumin, creatinine and 25-OH vitamin D.
Table 2 shows the analysed parameters in absolute values. The 4 In group B, mean serum CTX changes from baseline to week 4 were
gures present the changes between baseline, 2 weeks and 4 weeks in signicant (Fig. 1), as well as mean PTH changes (Fig. 2), while CTX and
the urine and serum parameters except for pH which was expressed in PTH stayed stable in group A. At week 4, the changes in serum CTX and
absolute values. PTH were signicantly different between the two groups.

Blood tests Urinary tests

The following parameters remained stable during the 4 weeks of 24 h urine

the trial and showed no signicant differences between the two The urinary volume increased regularly in both groups between
baseline and week 4 with no signicant difference between the two
groups.

Fig. 1. Average changes (+/SEM) of blood C-telopeptides according to water and study
week. Fig. 2. Average changes (+/SEM) of blood PTH according to water and study week.
 E. Wynn et al. / Bone 44 (2009) 120124 123

concentrations of uoride, chloride and phosphates content was

negligible. Therefore, none of these ions can inuence the results of
this study.

Bicarbonate

Bicarbonate and urine pH increased signicantly in Group B,

showing the expected impact of Water B on the acid-base metabolism.
The precision of the measurement of bicarbonate can be questioned as
the urine must be collected immediately after emission and
transported in an airless container. However, the results are
concomitant with the change in bicarbonate intake, as already
observed in a previous trial [20].
Fig. 3. Average (+/SEM) of pH in 24 h urinary excretion according to water and study
week.
Potassium

pH increased in group B between baseline and weeks 2 and 4 and The increase of potassium excretion on Water B cannot be
was signicantly different between both groups at 4 weeks (Fig. 3). explained by the K contents of the waters which were low and
Mean changes in urinary bicarbonate excretion between baseline similar, neither by the food intake which was controlled. It can be
and weeks 2 and 4 were signicant in group B and also signicant explained by the enhanced delivery of bicarbonate to the renal tubules
between both groups at week 4 (Fig. 4). which increases K excretion, the physiologic rationale being the
Creatinine and sodium excretion stayed stable and showed no possibility to eliminate more K [25].
signicant differences between the 2 groups. Calcium excretion
increased signicantly between baseline and week 4 in both groups Sulphate
(by 34.5 mg, SD 25.7 in group A p = 0.020, and by 27.7 mg, SD 12.8 in
group B p = 0.019), with no signicant difference between the groups. In general, calcium and sulphate contents in mineral water are
When expressed in percent of the absorbed calcium (calculated positively correlated [26]. One can argue that the sulphate content of the
according to reference 24), the urinary calcium excretion was 62k on two waters may play a role, since it was high in Water A. But in Water B,
Water A and 50k on Water B. Urinary CTX decreased slightly in both calcium is associated with bicarbonate but not with sulphate. In animals,
groups, but not signicantly, and no signicant differences between sulphate supplementation increases calcium excretion [18,27,28]. But
both groups were observed. Potassium excretion was signicantly studies in humans show contradictory results: sulphates either have no
different between both groups at week 4 (p = 0.033). It remained stable effect [29] or increase calciuria due to their acidogenic action [30].
in group A, but increased signicantly between baseline and week 4 in Indeed, acid load increases urine calcium excretion [30]. The acid load
group B (p = 0.0015). produced by the sulphate is included in the PRAL formula which shows
the total acid load. When applied to the two mineral waters, it was
Fasting morning urine estimated +9.2 mEq/l for water A (acid) and 11.2 mEq/l for water B
Expressed as ratio with creatinine, calcium, sodium, and CTX (alkaline). Therefore, the effects we observed were enhanced by the
stayed stable and showed no signicant differences between the two acidity of water A and the alkalinity of water B and were not exclusively
groups. Bicarbonate increased in group B throughout the 4 weeks and due to the difference in bicarbonate content.
was signicantly higher than in group A (p = 0.014). pH increased in
group B between baseline and week 4 (p = 0.0006) and was PTH
signicantly different between both groups at 4 weeks (p = 0.0001).
Potassium showed an increase in group B at week 2 (p = 0.014) and Serum PTH decreased on water B but not on water A. This was
week 4 (p = 0.013). surprising because total calcium, as well as ionized calcium remained
stable. The same phenomenon was observed in a previous human
Discussion study [8]. In dogs [31], metabolic alkalosis decreased PTH secretion
also without any change in blood calcium, supposedly by modifying
A 4-week dietetic intervention with 1.5 daily litres of calcium-rich the calcium-sensors on the parathyroid glands. In our study, the
mineral waters showed that additional bicarbonate enhances inhibi- calcium-rich water A did not lower the PTH level, perhaps because the
tion of bone resorption. The effect of calcium-rich mineral water on nutritional calcium intake was already relatively high at baseline. This
bone resorption has already been shown, but in postmenopausal, does not exclude a short-term effect [23], which however would not
calcium-decient women [3,23]. Our study was performed in calcium- inuence the blood parameters of the next morning. Therefore, the
and oestrogen-sufcient women.
Since all subjects followed the same diet plan, it can be assumed
that all differences observed were not inuenced by changes in
nutrition, but by the mineral waters only.

Calcium

The increase in urinary calcium excretion by approximately 30 mg/

24 h is in line with the increased calcium intake by 800 mg/d from the
mineral water. This is compatible with a fractional absorption rate of
about 20k at the given intake of calcium. When expressed in percent
of the absorbed calcium [24], the urinary calcium excretion was
signicantly lower (p b 0.001) in Group B, perhaps an indirect sign of a Fig. 4. Average changes (+/SEM) of bicarbonate in 24 h urinary excretion according to
decrease in bone resorption. Both waters had very low and similar water and study week.
124 E. Wynn et al. / Bone 44 (2009) 120124

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