Evaluation of the Effects of Bicarbonate-calcium Water in Premenopausal and Postmenopausal Women as a Prevention of Osteoporosis
Phase: NA
Conditions: Osteoporosis Risk
Sponsor: University of Salerno

STUDY PROTOCOL 
 
Official title: Evaluation of the effects of taking bicarbonate-calcium water in 
premenopausal and postmenopausal women as prevention of osteoporosis 
 
Date of Document: January 27th, 2021 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Study design: experimental study, non- pharmacological trial 
Background 
Adequate calcium supplementation in association with the Mediterranean diet and 
lifestyle physically active can improve the quality and even life expectancy in the 
population, with a reduced risk of osteoporosis. Mineral water can be a good source of 
calcium, 
highly bioavailable and easily assimilated. Calcium is naturally abundant in 
waters minerals that represent precious non-caloric sources. Mineral waters, 
assumed daily, with a high calcium content (over 150 mg/L) and a low sodium 
content 
(less than 20 mg/L) can be considered excellent sources of this mineral. 
Until a few years ago, calcium from water was believed to be poorly absorbable compared 
to that contained in other food sources, but several scientific studies have reversed this 
conviction, especially for calcium (calcium> 150 mg/L) and bicarbonate- calcium waters 
(bicarbonate > 600 mg/L). Furthermore, several studies have shown that the effects of 
sodium on the organism they are many and would also concern the bones, so much so that 
its effect hypercalciuric in the urine has led to hypothesize a possible role of sodium in the 
pathogenesis of osteoporosis. Bicarbonates make water alkaline and affect water markers 
bone resorption. In this scenario, the daily consumption of bicarbonate-calcium waters 
witha low sodium content is a major contributor to calcium intake and a 
significant protective factor for osteoporosis. 
Objective 
 
Evaluate the effectiveness of regular consumption of Lete water classified as bicarbonate- 
calcium e low sodium for the prevention of osteoporosis in premenopausal and postmenopausal 
women 
as part of a defined dietary protocol. 
Materials 
 
Subjects enrolled in the study: 
120 
women 
Inclusion Criteria: 
For premenopausal women age >40 years, for menopausal women period of onset of 
menopause1-10 years, with BMI of 19-30 Kg/m2 . 
 
 
 
1 

Exclusion criteria: 
 
Inclusion in other study protocols. Pharmacological therapies in progress: estrogen hormone 
therapy replacement, osteoporosis therapy, corticosteroid therapy, insulin therapy. 
Assumption of 
calcium supplements. Presence of moderate to severe kidney and liver 
disease, already documented severe osteoporosis condition (T-score MOC 
<-2.5). 
Waters: 
 
• Lete bicarbonate-calcium water, low in sodium 
 
• non-bicarbonate-calcium water: the ideal comparison water is low-mineral 
water from bottled with the same packaging as that of Lete water, with the 
addition of CO2. 
Methods 
 
The subjects enrolled for the study will be divided into two main groups: premenopause A 
andpostmenopausal B and then subsequently randomly in the two subgroups and 
respectively 
 
• Group A1 (intervention group): 30 premenopausal subjects who will drink water 
The bicarbonate-calcium Lete 
 
• Group A2 (control group): 30 premenopausal subjects who will drink water 
low mineral content 
• 
Group B1 (intervention group): 30 postmenopausal subjects who will drink water 
The bicarbonate-calcium Lete 
 
• Group B2 (control group): 30 postmenopausal subjects who will drink water 
 
low mineral content 
 
The study will be randomized single-blind. 
 
As for the water to be taken, the bottles will be delivered at the expense of the 
company SGAM SpA in the following ways: the entire study takes a total of 6 
months 
considering that the recruited subjects (120) must drink 2 L of water per day 43200 L of 
 
water, of which 21600 L Lethe bicarbonate-calcium water and 21600 L mineral water 
with addedCO2. For each subject, 240 1.5 L bottles are needed. As regards logistics, 

the subjects they will receive home delivery of the assigned water and precisely 10 
packs of 6 bottleseach for a total of 4 deliveries at set times. 
 
Sample size 
An analysis was performed to calculate the sample size and evaluate the power of the clinical 
trial statistics. The power of the (1-beta) test to be obtained, i.e., was considered as factors 
the probability of rejecting the null hypothesis when there is a real difference, the least difference 
between the treatments (control and intervention group), the variability of the measure and finally 
the level of significance alfa (5%), i.e. the probability of obtaining a statistically significant 
difference when in fact there is no difference. A priori information from the literature was used 
scientific. 
 
Study 
 
Study of the ability to assimilate calcium by drinking bicarbonate water with 
 
a high calcium and low sodium vs control (low mineral water) in women in pre 
 
and postmenopausal for a period of 6 months. After the end of the study there will be 
a period offollow-up for study monitoring. 
Subjects will follow a Mediterranean diet of 25-30 kcal/kg of ideal body weight/day 
(possibly free from acidic foods, controlling calcium intake 
<700 mg/day, of 1 g/weight ideal protein per day, carbohydrates with a low glycemic 
index 60%, lipids mainly of type 
unsaturated 40%). The effects of water intake will be evaluated in relation to the 
presence of bicarbonates also on the functionality of the digestive and locomotor 
systems. During recruitment i subjects will complete a FFQ Food Frequency 
Questionnaire to establish their daily intake 
of calcium in the diet. 
 
The patients will be recruited by the associations Amos Paternopoli, Amos Montoro, Amdos 
Forino,Amos Solofra, Noi in Rosa, Amos Valle del Sabato. 
The study includes measurements at 0, 3 and 6 months. Operational center of the study: 
Research LaboratoryUniversity nutrition Nutriketo_Lab, "San Giuseppe Moscati" Hospital of 
Avellino. 
 
The two groups will be subjected to the indicated study protocol and will carry out: 
 
• Recruitment test C (clinical, anthropometric, instrumental and blood chemistry tests) 
 
• Test I after set times (Mediterranean diet, with 2 L Lete water (30 women 
in group A1 and in group B1) or with 2 L mineral water (30 women in 
group A2 and in that 
B2), with 500 mL with meals three times a day and the remainder during the day. 
 
The following tests will be performed: 
 
Clinical examinations: Physiological and pathological history, physical examination and nutritional 

evaluation, 
 
to investigate the patient's clinical conditions and the functionality of the 
digestive and locomotive.systems 
Instrumental examinations: computerized bone mineralometry (MOC), articular ultrasound 
to evaluate the degree of elasticity, BIA/BIVA for the qualitative and quantitative evaluation 
of the body composition; 
Metabolomic analysis of serum, urine, and saliva samples by spectroscopy nuclear magnetic 
resonance to investigate change in glycemic, lipidic and bone metabolism. 
 
Processing of results 
 
 
Statistical analysis, data visualization and predictive analysis will be carried 
out through Statgraphics software (Statgraphics Technologies, Inc., The 
Plains, Virginia). Features of the population included in the study will be 
analyzed by descriptive techniques. In 
all evaluations made the results will be expressed as arithmetic mean and deviation 
 
standard. The statistical analysis of the parametric data will be performed with the Student's T 
test for measurements paired and unpaired, comparing data at the start and at the end and then 
between the control and the intervention group. P-values <0.05 will be considered statistically 
significant. 
 
Study monitoring 
 
The study will be performed in accordance with the protocol and international Good 
Clinical guidelines Practice, and with the current regulations on clinical trials and those 
of reference for 
natural mineral waters. 
 
Ethical aspects 
 
The current version of the Declaration of Helsinki (2013), the reference for the 
ethical aspects of this clinical trial will be respected by all who are engaged in this 
research. 
The protocol, informed consent and any other material will be reviewed by the 
Ethics Committee. 
 
All eligible subjects must be informed of the purpose of the study, possible risks, and 
 
benefits of the proposed interventions, and must obtain an information document stating 
the nature,the purpose and course of the study. 
The data of the study subject will be confidential and used in accordance with current 
legislation onprotection of sensitive data and privacy legislation. 
Outcomes 
 
The intake of an easily assimilable dose of calcium from a resource such as bicarbonate 
water calcium and sodium levels, may be able to determine 

improvements in turnover markers 
bone and also joint functionality and elasticity, decreasing the risk of bone fractures. 
 
 
Study investigator: Prof. Luca Rastrelli, Full Professor of Food Chemistry, 
 
Department of Pharmacy, University of Salerno, Via Giovanni Paolo II, 
132, 84084 Fisciano SA- Nutritional research laboratory Nutriketo_Lab, 
San Giuseppe hospital Moscati, Contrada Amoretta, 83100 Avellino AV 
Doctor: Dr. Giuseppe Castaldo, Scientific Director of the Research Laboratory 
 
nutritional Nutriketo_Lab, San Giuseppe Moscati hospital, Contrada 
Amoretta, 83100Avellino Av 
Address: Nutritional research laboratory Nutriketo_Lab, Company 
 
San Giuseppe Moscati hospital, Contrada Amoretta, 83100 Avellino AV 
 
Study sponsor: SGAM SpA 
 
 
 
 
Bibliography 
 
• Böhmer, H., Müller, H., & Resch, KL (2000). Calcium supplementation with 
calcium-rich mineral waters: a systematic review and meta-analysis of its 
bioavailability. Osteoporosis international,11(11), 938-943 . 
• Burckhardt, P. (2008). The effect of the alkali load of mineral water on 
bone metabolism: interventional studies. The Journal of nutrition, 
138(2), 435S-437S. 
• Cepollaro, C., Orlandi, G., Gonnelli, S., Ferrucci, G., Arditti, JC, Borracelli, D., 
... & Gennari, C. (1996). Effect of calcium supplementation as a high-calcium 
mineral water on bone loss in early postmenopausal women. Calcified tissue 
international, 59(4), 238-239. 
• Guéguen, L., & Pointillart, A. (2000). The bioavailability of dietary 
calcium. Journal of the American College of Nutrition, 19(sup2), 119S- 
136S. 
• Guillemant, J., Le, HT, Accarie, C., du Montcel, ST, Delabroise, AM, Arnaud, MJ, & 
 
Guillemant, S. (2000). Mineral water as a source of dietary calcium: acute effects on 
parathyroid function and bone resorption in young men. The American journal of clinical 
nutrition, 71(4), 999- 
1002. 
 
• Heaney, RP, & Dowell, MS (1994). Absorbability of the calcium in a high-
calcium mineralwater. Osteoporosis International, 4(6), 323-324. 
• Panel, CD (1994). Optimal calcium intake. NIH Consensus Conference. JAMA, 272, 1942- 
1948. 

• Porter, DV (1994). Washington update: NIH consensus development conference 
statement optimalcalcium intake. Nutrition Today, 29(5), 37-40. 
• Quattrini, S., Pampaloni, B., & Brandi, ML (2016). Natural mineral waters: 
chemical characteristics and health effects. Clinical Cases in Mineral and 
Bone Metabolism, 13(3), 173. 
• Vannucci, L., Fossi, C., Quattrini, S., Guasti, L., Pampaloni, B., Gronchi, G., 
... & Brandi, ML (2018). Calcium Intake in Bone Health: A Focus on 
Calcium-Rich Mineral Waters. nutrients,10(12), 1930. 
 
• Wynn, E., Krieg, MA, Aeschlimann, JM, & Burckhardt, P. (2009). Alkaline mineral water 
lowers bone resorption even in calcium sufficiency:: Alkaline mineral water and bone 
metabolism. 
Bone, 44(1), 120-124. 
 
 
 
 
 
Laboratory tests will be performed on 120 patients at 0, 3 and 6 months. 
• Vitamin D 
• Albumin 
• Bicarbonates 
 
• Football (S) 
• Football (U) 
• Ionized Calcium 
• Creatinine -S/U/DU/LA- 
• Creatinine clearance 
 
• Emocrome 
• Urine test 
 
• Alkaline phosphatase (ALP)(S) 
 
• Phosphorus (S) 
 
• Phosphorus (U) 
• Magnesium S/U/DU/(SG)ER- 
• Osteocalcin* 
 
• P1NP N-terminal propeptide of type I procollagen (S) 
 
• Parathyroid hormone (PTH) 

• Sodium S/U/DU/(SG)ER- 
• C-terminal collagen telopeptide (CTX) (S) 
• Total 
cholesterol 
• HDL 
• LDL 
 
 
 
The MOC exam (Computerized Bone Mineralometry) will be performed on 120 patients at time 0 
and 
 
6 months.