The aim of the present study was to elucidate how bone metabolism and bone mineral density are affected by the consumption of a lemon juice-containing calcium (Ca)-enriched beverage. The efficacy of this investigational product was evaluated in postmenopausal women during five months of continuous intake (intervention). This was a randomized, controlled trial. Eighty-three subjects were assigned to three groups. Using a double-blind format, the first two groups received a Ca-supplemented lemon-juice (lemon) beverage (LECA) or a Ca-unsupplemented lemon-juice (lemon) beverage (LE). The third group (control) received no intervention. Each subject in the LECA and LE groups consumed one bottle (290 mL) of their assigned investigational product every day for five consecutive months. After five months of intervention, the gain in bone mineral density at the lumbar spine was significantly larger in the LECA and LE groups than in the control group. In the femur, subjects in the LECA group gained significantly more bone mineral density than the control subjects. The largest gain in bone mineral density at the lumbar spine was observed in the LECA group. As for the concentrations of the bone resorption marker tartrate-resistant acid phosphatase 5b (TRACP-5b), subjects in the LECA group had significantly lower values than those in the control group. Similarly, when compared with the LE and control groups, a significant decrease was detected in the LECA group in the concentrations of the bone formation markers, bone alkaline phosphatase (BAP) and osteocalcin (OC). In postmenopausal women, continuous consumption of Ca-supplemented lemon beverages improved the absorption of Ca and inhibited bone resorption. This likely blocked the function of osteoblasts and led to the suppression of bone formation, resulting in the attenuation of high-turnover bone metabolism.
Osteoporosis and the subsequent risk of bone fracture are characterized by enhanced bone fragility, resulting in an increased risk of fracture, and it is usually defined as a reduction in bone mineral density (BMD) [
Citric acid is mainly responsible for the acidic flavor of lemons. Recent studies have demonstrated that this organic acid acts as a chelating agent and promotes the absorption of minerals such as Ca and iron [
In this study, the aim was to elucidate how bone metabolism and BMD are affected by the consumption of a lemon juice-containing Ca-enriched beverage. Using a double-blind experimental design, the efficacy of the beverage was examined in postmenopausal women during five-month continuous consumption (intervention).
Volunteer subjects were recruited by M city public advertising. A participation condition is to be being normal and a postmenopausal woman, and the person that the ovary and the uterus which have an influence on the osteoporosis do not have a history of treatment. Exclusion criteria are the person with a history of the serious disease and the person whom it was judged to be inappropriate by the investigator. They were given detailed information about the study (such as aims and methods) at an orientation session offered prior to enrollment in the study. Written, informed consent was obtained from 97 volunteers. Of these, 83 were selected for statistical analysis. They had undergone all scheduled examinations conducted at the beginning of the study (prior to intervention with investigational products), as well as two and five months after study initiation.
This was a randomized, controlled trial. An identification number was assigned to each subject for anonymization. Subjects were divided into three groups matched for age and body mass index (BMI). Each group underwent a different intervention. In the LECA group, subjects received a Ca-supplemented lemon-juice (lemon) beverage. In the LE group, subjects received a Ca-unsupplemented lemon-juice (lemon) beverage. The third group (control) received no intervention. The placebo was not used in this study. The control group lived a usual life. The investigational products were distributed by Pokka Sapporo Food & Beverage Ltd. using a double-blind protocol. This study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of the Prefectural University of Hiroshima (Approval Number 15MH025-01).
The study was conducted between December 2015 and June 2016. Each subject in the LECA and LE groups consumed one serving (290 mL) of an investigational product every day for five consecutive months. Assessments of physical measurements, BMD, and bone metabolism markers were performed at study initiation (pre-intervention) and after two and five months of intervention.
Age and the number of years since menopause were obtained by interview. Physical measurements consisted of determining height, weight, and BMI.
BMD (g/cm2) values of the lumbar spine (L2-L4) and left femur were determined using the X-ray bone densitometer Discovery (Toyo Medic Co., Ltd., Tokyo, Japan). During the course of this study, the densitometer was calibrated 99 times with the following results: mean = 1.038 g/cm2, SD = 0.003 g/cm2, and CV = 0.37%. These values were within the normal ranges established by the manufacturer, Hologic, Inc. (Marlborough, MA, USA).
Bone Resorption Markers: The concentrations of serum TRACP-5b and urinary type I collagen cross-linked N-telopeptide (u-NTx) were measured.
Bone Formation Markers: The concentrations of bone alkaline phosphatase (BAP) and osteocalcin (OC) were measured. Blood samples collected from study subjects were centrifuged at 1500× g for 10 min to obtain serum for analyzing TRACP-5b, BAP, and OC concentrations. The analysis was carried out using commercially available kits. Similarly, u-NTx was detected in urine from subjects using a commercially available kit, without sample pretreatment. All serum and urine assays were performed by Fukuyama Medical Laboratory Inc. (Hiroshima, Japan). The kits used for these assays were: Osteolinks “TRAP-5b” (Nittobo Medical Co., Ltd., Tokyo, Japan) for serum TRACP-5b; Access Ostase (Beckman Coulter Co., Ltd., Brea, CA, USA) for BAP; BGP IRMA “LSI M” (LSI Medience Co., Ltd., Tokyo, Japan) for OC; and Osteomark (Alere Medical Co., Ltd., Tokyo, Japan) for u-NTx.
Data obtained prior to intervention are presented as means ± standard deviation. After adjusting for age and BMI, the correlations between investigational products and intervention periods were analyzed by two-way analysis of variance. The two independent variables were intervention groups (LECA, LE) or no-intervention group (control), and intervention duration [intra-individual levels at study initiation (pre-intervention), at two months of intervention, or at five months of intervention]. The dependent variable was BMD (at the lumbar spine or femur). The correlations of intervention periods with investigational products and the levels of bone metabolism markers were also analyzed using the same statistical technique and the identical independent variables. The dependent variables were the concentrations of bone metabolism markers. The normality of the distributions of BMD values and bone metabolism marker levels was assessed by histograms and the Kolmogorov-Smirnov test (p = 0.200). For each subject group, the net gain in BMD was determined by subtracting the pre-intervention BMD from the BMD detected after five months of intervention.
LECA Group | LE Group | |
---|---|---|
Name | Ca-supplemented lemon-juice (lemon) beverage | Ca-unsupplemented lemon-juice (lemon) beverage |
Intake Amount | 290 mL/bottle, 1 bottle/day | |
Composition | Per bottle (290 mL) Calcium 350 mg Lemon juice 30 mL Calorie 49 kcal | Per bottle (290 mL) Calcium 0 mg Lemon juice 30 mL Calorie 20 kcal |
Dosage and Administration | 1 bottle/day. Any time of the day |
The obtained values were then analyzed by one-way analysis of variance, followed by the Dunnett multiple comparison test. The level of significance for all tests was set at p < 0.05.
There were no significant differences in the BMD of the lumbar spine among the means of the three subject groups (p = 0.541) or between different time points (p = 0.902). However, there was a significant interaction effect (p < 0.001) (
Similarly, there were no significant differences in the BMD of the femur among the means of the three subject groups (p = 0.709) or between different time points (p = 0.807). Again, the interaction effect was significant (p < 0.001) (
Clinical Variable | Intervention Group 1 (n = 28) Mean (SD) Min - Max | Intervention Group 2 (n = 28) Mean (SD) Min - Max | Control Group (n = 27) Mean (SD) Min - Max | Total (n = 83) Mean (SD) Min - Max |
---|---|---|---|---|
Age (years) | 65.2 (5.2) 57.0 - 82.0 | 65.1 (5.7) 56.0 - 81.0 | 64.1 (6.7) 48.0 - 80.0 | 64.8 (5.8) 48.0 - 82.0 |
Age at Menopause (years) | 48.7 (5.8) 35.0 - 60.0 | 49.5 (5.6) 30.0 - 55.0 | 51.4 (2.4) 46.0 - 55.0 | 49.6(4.8) 30.0 - 60.0 |
Height (cm) | 152.8 (5.6) 143.2 - 164.1 | 154.4 (4.7) 147.5 - 162.5 | 154.0 (4.7) 141.1 - 162.8 | 153.7†(5.1) 141.1 - 164.1 |
Baseline Weight (kg) | 52.3 (7.2) 40.3 - 65.7 | 52.9 (8.5) 41.9 - 72.9 | 52.4 (11.0) 30.3 - 79.5 | 52.5‡ (8.9) 30.3 - 79.5 |
Baseline BMI (kg/m2) | 22.4 (2.8) 17.8 - 29.0 | 22.4 (2.8) 17.8 - 29.0 | 22.1 ( 4.5) 13.3 - 34.5 | 22.2 (3.6) 13.3 - 34.5 |
†: vs 153.4 (4.8), n = 2444, by Health and Welfare Statistics Association, p = 0.192271; ‡: vs 53.9 (7.8), n = 2443 by Health and Welfare Statistics Association, p < 0.001.
TRACP-5b concentrations did not show any significant differences among the means of the three subject groups (p = 0.624) or between different time points (p = 0.331). However, the interaction effect was significant (p < 0.001) (
As for the concentrations of OC, there were no significant differences among the means of the three subject groups (p = 0.835) or between different time points (p = 0.328). The interaction effect was significant (p = 0.012) (
Finally, BAP concentrations did not show any significant differences among the means of the three subject groups (p = 0.234) or between different time points (p = 0.138), although the interaction effect was significant (p < 0.001) (
and between the LECA and control groups were both significant (both p < 0.001) (
In this study involving postmenopausal women, there was a significant interaction effect for the concentration of the bone resorption marker TRACP-5b. When the pre-intervention TRACP-5b level was subtracted from the TRACP-5b level after five months of intervention, a negative value was obtained for the LECA group. This value differed significantly from the positive values obtained by the same formula for the LE and control groups. Significant interaction effects were also observed for the concentrations of the bone formation markers OC and BAP. The value obtained by subtracting the pre-intervention OC level from the OC level after five months of intervention was negative for all three subject groups. However, the differences in these values between the LECA and LE groups and between the LECA and control groups were both significant. As for BAP, the value obtained by subtracting the pre-intervention level from the level after five months was negative for the LECA group and positive for the LE and control groups. The differences in these values between the LECA and LE groups and between the LECA and control groups were both significant. The results of previous studies that evaluated the effects of dietary Ca intervention are controversial. Matsumoto et al. [
The increase in the BMD of the lumbar spine was calculated for each subject group by subtracting the pre-intervention value from the value obtained after five months of intervention. The results indicated that there was a significant difference in the increase between the LECA and control groups. A similar result was obtained for the BMD of the femur. Bone is constantly renewed through a process called remodeling [
Moschonis et al. [
Since estrogen deficiency causes postmenopausal osteoporosis, inhibitors of bone resorption (such as estrogen and bisphosphonate preparations) have been used for the prevention and treatment of osteoporosis. In fact, Wallach et al. [
In this study, a survey on individual diet remains to be done. Nevertheless, a new experimental result was achieved by ingesting the test beverage while having a normal life.
The physical measurements of each subject selected for the present analysis were similar to the mean values obtained from an age-matched Japanese population [
In postmenopausal women, continuous consumption of Ca-supplemented lemon beverages improved Ca absorption and inhibited bone resorption. The suppression of bone resorption likely blocked bone formation mediated by the proliferation and differentiation of osteoblasts, resulting in the attenuation of high-turnover bone metabolism. The novel finding of the present study was that this attenuation was reflected in BMD. The present results also suggest that citric acid in lemons enhances the absorption of dietary Ca. We expect that such beverages will have an efficacy in preventing osteoporosis in the future.
The authors are grateful to the volunteer of the M city which participated in this study. We also thank Prof. H. Miyaguchi of Hiroshima University and Prof. Y. Nitta of Hiroshima Shudo University for experiment assistance with this study.
The authors declare no conflicts of interest regarding the publication of this paper.
Ikeda, H., Iida, T., Hiramitsu, M., Inoue, T., Aoi, S., Kanazashi, M., Ishizaki, F. and Harada, T. (2018) Effects of Lemon Beverages on Bone Metabolism and Bone Mineral Density in Postmenopausal Women: A Double-Blind, Controlled Intervention Study with Ca-Supplemented and Unsupplemented Lemon Beverages. Open Journal of Preventive Medicine, 8, 301-314. https://doi.org/10.4236/ojpm.2018.810026