Efficacy of Potassium Citrate in the Treatment of Postmenopausal Osteopenia. A Randomized, Placebo-controlled, Double Blind Investigation.
Phase: NA
Conditions: Osteopenia, Bone Disease, Metabolic
Sponsor: Istituto Ortopedico Rizzoli

NCT02731820    Efficacy of Potassium Citrate in the Treatment of Postmenopausal Osteopenia 6013_IOR 
 
 
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Efficacy of Potassium Citrate in the 
Treatment of Postmenopausal 
Osteopenia 
 
Approval:  9 March 2016 
 
 
 
 
 
Prof. Nicola Baldini 
SSD Laboratorio di Fisiopatologia Ortopedica e Medicina Rigenerativa  
IRCCS Istituto Ortopedico Rizzoli 
 
 

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Study protocol and statistical analysis plan 
 
1. Study Design and Participants 
A randomized, double-blind, placebo-controlled, parallel-group study, with balanced randomization 
(1:1) was designed. The study was approved by the local research committee and registered at Clinical 
Trial Gov (ID: NCT02731820). The participants were recruited from a cohort of postmenopausal 
women treated at the Radiodiagnostic Unit of Rizzoli Orthopedic Institute (IOR) from January 2016 
to June 2017; periodic measurements of lumbar (at the L2–L4 level) and femoral BMD were carried 
out using dual-energy X-ray absorptiometry (DXA) (Discovery DXA System, Hologic Inc., Bedford, 
MA, USA). Written informed consent was obtained from all participants. A code was assigned to 
each woman at the beginning of the trial which was used to track the data throughout the study. The 
inclusion criteria were women with osteopenia (T-score <−1 and >−2.5) and more than 5 years post-
menopause. The risk of fracture was evaluated by using the FRAX® tool [1] based on an algorithm 
that takes into account demographic characteristics, medical history, some clinical risk factors as well 
as bone mineral density (BMD) at the femoral neck. The exclusion criteria were risk of fracture 
(FRAX: >20 major osteoporotic fractures; >3 hip fractures), hyperkalemia, renal insufficiency, 
urolithiasis, use of therapies influencing bone metabolism (e.g., corticosteroids, aromatase inhibitors 
and estrogens), use of drugs (such as diuretics and proton pump inhibitors), calcium and potassium 
supplements, vitamin D, current or recent (less than three years prior to the start of the study) use of 
bisphosphonates, gastrointestinal disorders which hamper nutrient absorption, and mental or 
psychiatric disorders which precluded the possibility of correctly adhering to the protocol. All the 
women were interviewed regarding food habits; they followed their usual diet throughout the duration 
of the study. Participants were assigned to the treatment or the placebo group using a randomization 
sequence created by independent personnel (IOR pharmacy) using a free online tool 
(http://randomization.com) and were stratified with a 1:1 allocation using a random block size of 4. 
 
2. Supplements 
The K citrate and the placebo were kindly provided by a manufacturer (Biohealth, Turin, Italy). They 
were in tablet form, identical in appearance, and were packaged in bottles which were rendered 
anonymous (A and B) until the end of the study. The products were stored at the IOR pharmacy and 
dispensed according to the computer-generated randomization list. Each woman received the 
supplements in the corresponding prepacked bottle. Both the subjects and the investigators who 
followed up the participants were blinded to group assignment. All subjects had daily 
supplementation with vitamin D3 cholecalciferol (400 IU day−1) and calcium carbonate (500 mg 

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day−1). The participants assigned to the experimental group received 30 mEq K citrate daily in two 
tablets for oral administration; those assigned to the placebo group received the same quantity of 
tablets containing only the excipients. The participants had clinic visits at the time of randomization 
(baseline), and at 3 and 6 months. At each time point, they underwent blood and urine evaluations. 
The laboratory staff performing the assays was unaware of the source of the samples which were 
identified by numeric codes. 
 
3. Blood Analyses 
Fasting venous blood samples were collected and analyzed for creatinine, sodium, potassium, total 
calcium, inorganic phosphate, parathyroid hormone (PTH), and 1,25-dihydroxyvitamin D. These 
analytes were measured by routine methods. 
 
4. Urine Analyses 
Urine samples were collected using a standardized protocol (Lithotest®, Biohealth, Turin, Italy), and 
the metabolic profile was examined in an external certified lab (Lithocenter, Turin, Italy). Briefly, 
during collection, 24-hour (24 h) urine was aliquoted into two fractions to which 10 mL hydrochloric 
acid (to prevent calcium/phosphate precipitation) and 1 mL Hibitane (to prevent bacterial 
contamination) were added. The acidified aliquot was used to quantify calcium (Ca), citrate, 
magnesium (Mg), oxalate, phosphate (PO4) and sulphate (SO4); the Hibitane aliquot was tested for 
ammonium, chloride (Cl), creatinine, pH, potassium (K), sodium (Na), urea, and urate. In addition, a 
sample of fasting-morning urine was collected within 2 h after the 24 h collection ended, and was 
analyzed for citrate, calcium, urate (all expressed as creatinine ratios) and pH. Reference values were 
established on the basis of the assessment of mineral metabolic profile to evaluate the lithogenic risk 
[2,3,4]. The renal acid load was determined by calculating the 24 h urinary “potential renal acid load” 
(uPRAL) as follows [5]: 
[Cl (mmol day−1) + SO4 (mmol day−1) × 2 + PO4 (mmol day−1) × 1.8] − [Na (mmol day−1) + K (mmol 
day−1) + Mg (mmol day−1) × 2 + Ca (mmol day−1) × 2] 
 
5. Immunoenzymatic Assay of BTM 
The serum level of the bone turnover markers (BTMs) was evaluated at baseline and after 3 and 6 
months of treatment. The BTM assay was carried out by following the recommended actions to 
minimize the pre-analytical variability [6]. The blood samples were collected after overnight fasting 
(between 08:00 and 10:00 a.m.), centrifuged within 2 h from collection, at 1000 g min−1 for 15 min 
and at room temperature. The serum was transferred into cryotubes (0.5 mL of serum per cryotube) 

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and frozen at −80 °C, within 1 h after centrifugation, until testing. The BTM serum levels were 
measured using commercially available reagents based on a sandwich enzyme immunoassay 
technique, following the manufacturer’s protocols, with each sample tested in duplicate. The selected 
BTMs were related to (i) the number of osteoclasts (tartrate-resistant acid phosphatase 5b; 
TRACP5b); (ii) osteoblast function (bone alkaline phosphatase; BAP); (iii) type I collagen 
degradation (carboxy-terminal telopeptide; CTX); (iv) type I collagen precursor (procollagen type 1 
N terminal propeptide; PINP). Source and technical notes of reagents used in this study are shown in 
Table 1. 
 
Table 1. Source of the immunoenzymatic assay kits, sensitivity, precision, least significant change (LSC) and references 
values from healthy individuals.  
Marker (Unit) 
Commercial name 
(Source) 
Detection 
limit 
Min - Max 
intraassay CV 
(%) 
Min - Max 
interassay CV 
(%) 
LSC  
(%)  
Mean ± SEM 
Median 
Min - Max 
 
 
 
 
 
Female 
(postmenopausal) 
Female 
(premenopausal) 
Male 
 
CTX (µg · L-1) 
Serum Crosslaps - 
(Immunodiagnostic 
systems Limited, 
Boldon, UK) 
0.02 
1.8 - 3.0 
2.5 - 10.9 
16.6 
0.64 ± 0.36 
0.44 
0.12-1.35 
0.38 ± 0.19 
0.29 
0.11-0.74 
0.39 ±0.19 
0.29 
0.12-0.75 
BAP (µg · L-1) 
Ostase BAP - 
(Immunodiagnostic 
systems Limited) 
0.7 
2.6 - 6.5 
3.7 - 6.1 
15.6 
14.53 ± 4.21 
13.2 
8.0-22.4 
9.0±2.98 
8.78 
4.0-14.30 
13.13±3.8 
12.3 
7.0-20.1 
PINP (pg · L-1) 
Human Procollagen I 
N-terminal Peptide  
(Cusabio Technology 
LLC, Aachen, 
Germany) 
18.75 
8 
10 
29.8 
22.6 ± 1.7 1 
20.1 
15.4–40.8 
TRACP5b (U · L-1) 
Bone TRAP 
(Immunodiagnostic 
systems Limited) 
0.5 
6.0-6.6 
5.8-7.2 
21.1 
3.19±0.85 
2.59±0.78 
3.06±0.88 
1 Reference values have not been differentiated by gender and age. 
 
6. Calculations and Statistical Analysis 
All calculations and analyses were carried out using StatView 5.01 for Windows (SAS Institute Inc., 
Cary, NC, USA) and MedCalc Statistical Software version 18.2.1 (MedCalc Software bvba, Ostend, 
Belgium). The sample size calculation was based on a previous randomized clinical trial (RCT) aimed 
to evaluate the effect of K citrate on calcium metabolism and BTM in a cohort of individuals (males 
and females, age >55 years) treated for 6 months [7]. The present RCT was powered considering a 
Type I error (α) = 0.05 (two-sided), and Type II error (β) = 0.20 (power is 80%). Fifteen patients per 
group were sufficient, but the expected proportion of dropouts was 20%; therefore, as a precaution, 
20 women in each group were recruited. Quantitative data were expressed as the arithmetic mean plus 
or minus the standard error of the mean (mean ± SEM). The D’Agostino-Pearson method was used 

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to test the normality assumption of continuous variables, and a log transformation was applied when 
the data distribution was non-normal. The data analysis was carried out following intention-to-treat 
principles. All follow-up parameters were analyzed using a “last value carried forward” approach 
under the intention-to-treat principle in order to address missing data [8]. The effect of K citrate on 
BTMs and blood/urine analytes was evaluated by applying the t-test, with comparison within the 
groups (paired analysis of the effect of treatment over time), and between the groups (independent 
comparison between K citrate and the placebo at the same time point). In order to highlight the 
changes over time, the results were expressed in line-graphs as deviations from the value observed 
before treatment, i.e.,  [(BTM concentration at time point × BTM concentration at baseline−1) − 1] 
Thus, obtaining a baseline value equal to zero. The degree of association between the continuous 
variables was calculated by measuring the Pearson correlation coefficient. Differences and 
correlations were considered to be statistically significant when the p value was <0.05. 
 
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