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.
2.2.4. Investigational Products
Table 1 shows the amounts of principal ingredients in the products tested in this study. The recommended dietary reference intake for Ca for adult women is set at approximately 700 mg per day  . In the present study, one-half of this value (i.e., 350 mg) was used as the intake amount. The amount of lemon juice in the investigational products was determined by referring to the health survey conducted by Domoto et al.  . They found that, in a lemon-growing region of Hiroshima Prefecture, individuals with high daily lemon consumption (approximately 30 mL, on average) had significantly lower blood pressure than those with low consumption. Based on this observation (the daily consumption amount was approximately 30 mL on average in the high daily lemon consumption group), the lemon juice amount was set to 30 mL. The amounts of Ca and lemon juice in the investigational products were within the ranges of values found in normally consumed foods. There have been no specific reports indicating that they can potentially pose health hazards. Thus, they were deemed safe.
2.2.5. Data Analysis
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.
Table 1. Composition of investigational products and their dosage and administration.
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.
3.1. Comparison of Baseline Physical Characteristics
Table 2 shows the baseline physical characteristics of the study subjects. There were no significant differences in any of these variables among the three subject groups.
3.2. Changes in BMD
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) (Figure 1(a)). When the pre-intervention BMD of the lumbar spine was subtracted from the BMD after five months of intervention, positive values were obtained for the LECA and LE groups, while a negative value was obtained for the control group. The differences in these values between the LECA and control groups and between the LE and control groups were both significant (both p < 0.001) (Figure 1(b)).
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) (Figure 1(c)). The value obtained by subtracting the pre-intervention BMD from the BMD after five months of intervention was positive for the LECA group. For the LE and control groups, the values obtained by the same method were negative. The difference in these values between the LECA and control groups was significant (p = 0.036). The difference between the LE and control groups was borderline significant (p = 0.064) (Figure 1(d)).
Table 2. Physical characteristics of study subjects.
†: 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.
(a) (b) (c) (d)
Figure 1. Relationship between investigational products and BMD. (a) BMD of the lumbar spine; (b) Changes in BMD of the lumbar spine; (c) BMD of the femur; (D) Changes in BMD of the femur. BMD: Bone Mineral Density. (a, c) Figures showed mean and SD error bar, and dotted line is control group, dashed line is LE group, solid line is LECA group. Two-way factorial analysis of variance with the kind of investigational products and intervention period as factors. In this result, they showed each of p value, investigational products are “Drink”, intervention period is “Time”, and interaction is “Interaction”. (b, d) Figures showed the results obtained by the Dunnett multiple comparison test after analyzed by one-way analysis of variance of each subject group the margin between five months of intervention and the pre-intervention.
3.3. Changes in the Concentrations of Bone Metabolism Markers
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) (Figure 2(a)). 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, whereas positive values were obtained 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 (both p < 0.001) (Figure 2(b)). In the case of u-NTx, its concentrations did not differ significantly among the means of three subject groups or between different time points. There was also no significant interaction effect (Figure 2(c)). Furthermore, there were no significant differences in u-NTx levels before and after the five-month intervention (Figure 2(d)).
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) (Figure 3(a)). When the pre-intervention OC level was subtracted from the OC level after five months of intervention, a negative value was obtained for all subject groups. However, the value obtained for the LECA group was significantly smaller than that obtained for the LE group (p = 0.008) or control (p = 0.033) group (Figure 3(b)).
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) (Figure 3(c)). The value obtained by subtracting the pre-intervention BAP level from the BAP level after five months of intervention was negative for the LECA group. Conversely, the same calculation yielded positive values for the LE and control groups. The differences in these values between the LECA and LE groups
(a) (b) (c) (d)
Figure 2. Relationship between investigational products and bone resorption markers. (a) Concentrations of TRACP-5b; (b) Changes in TRACP-5b concentrations; (c) Concentrations of u-NTx; (d) Changes in u-NTx concentrations. TRACP-5b: tartrate-resistant acid phosphatase type 5b. u-NTx: urinary type I collagen cross-linked N-telopeptide. The explanations in the figures of (a, c) and (b, d) are similar to figure legends of Figure 1.
(a) (b) (c) (d)
Figure 3. Relationship between investigational products and bone formation markers. (a) Concentrations of OC; (b) Changes in OC concentrations; (c) Concentrations of BAP; (d) Changes in BAP concentrations. OC: osteocalcin. BAP: bone alkaline phosphatase. The explanations in the figures of (a, c) and (b, d) are similar to figure legends of Figure 1.
and between the LECA and control groups were both significant (both p < 0.001) (Figure 3(d)).
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.  found significant decreases in bone resorption markers, whereas Kuwabara et al.  identified no significant changes. These discrepancies are partly due to differences in age between study participants. In the report by Kuwabara et al.  , 68 institutionalized elderly subjects consumed a jelly containing Ca (200 mg) and vitamin D3 (800 IU) every day for one month. After study completion, the authors found no significant changes in the concentrations of TRACP-5b and BAP, although serum 25(OH)D levels increased significantly. Separately, Toba et al.  examined the effect of consumption of the milk basic protein (MBP), a protein assembly with basic isoelectric points, on bone metabolism markers in adult men (mean age 36.2 years). They reported that, after a 16-day intervention, the concentrations of OC and u-NTx showed a significant increase and decrease, respectively. These were both short-term studies, with intervention periods of one month or less. In the present study, each subject regularly received an investigational product for five months, and, upon study completion, subjects in the LECA group had significantly lower levels of both bone resorption and bone formation markers. We hypothesize that when Ca-supplemented lemon beverages are regularly consumed, citric acid promotes the absorption of this mineral through its chelating activity. This would help maintain blood Ca concentrations within the normal range, resulting in lower levels of bone resorption and bone formation markers. In the LECA group, normal levels of blood Ca presumably eliminated the need for supplying this mineral from bone and suppressed osteoclast-mediated bone resorption. This likely inhibited the function of osteoblasts and led to the attenuation of bone formation  .
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  . This process elegantly regulates the balance between osteoclastic and osteoblastic activity, thereby preserving dynamic bone homeostasis. In other words, normal bone tissue is maintained through the resorption of an appropriate amount of old bone and the subsequent formation of new bone  . Changes in bone metabolism are induced during menopause, resulting in high bone turnover in which both bone resorption and bone formation are enhanced. However, bone resorption rates are much higher than bone formation rates, thus causing a reduction in bone mass   . In the present study, the maintenance of blood Ca levels likely inhibited bone resorption. This must have suppressed the proliferation and differentiation of osteoblasts and caused the attenuation of high-turnover bone metabolism.
Moschonis et al.  observed a significant increase in the BMD of the lumbar spine in postmenopausal women who had received a 12-month intervention consisting of lifestyle counseling and the consumption of dairy products. Aoe et al.  also reported a similar observation in healthy postmenopausal women who had consumed MBP for six months. In the present study, the novel finding was that the changes in bone metabolism markers caused by the regular consumption of Ca-supplemented lemon beverages were reflected in BMD levels.
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.  found that risedronate treatment significantly increased or maintained BMD of the lumbar spine and femur in patients receiving corticosteroid therapy. Furthermore, according to a report by Yoshida et al.  , serum NTx levels were greatly reduced in early postmenopausal women within one month following six-month raloxifene treatment. The authors also mentioned the inhibitory effect on bone resorption. However, while estrogen-based hormone replacement therapy can reduce the risk of fracture and colon cancer, it reportedly increases the risk of breast cancer, thrombosis, coronary artery disease, and stroke  . In addition, although bisphosphonate preparations can increase bone mass and prevent bone resorption and fractures, their dosing instructions are not easy to follow. An example is that, upon arising, patients need to swallow bisphosphonate tablets whole with a full glass (approximately 180 mL) of water on an empty stomach, without lying down and consuming anything other than water for at least 30 min afterwards. Thus, one of the disadvantages of bisphosphonate therapy is poor patient compliance. Indeed, several groups have previously shown suboptimal adherence to daily oral bisphosphonate therapy    . Another problem associated with the pharmacological intervention of osteoporosis is that an individual must be diagnosed with this disease (i.e., BMD < 70% of the young adult mean) to receive a prescription for bisphosphonate. Considering the importance of preventive medicine in the management of osteoporosis, it would be essential to develop an alternative to conventional osteoporosis drugs that would be effective even for individuals whose BMD is 70% or higher. One can drink the Ca-supplemented lemon beverage investigated in this study regardless of the presence or absence of a disease or diagnosis. There are no instructions or restrictions as to when or how the beverage should be taken. Thus, it can be easily consumed regularly for an extended period of time.
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  . There were no significant difference between the average height of the subjects and Japanese women in their age group, but the subjects were below average in weight. This difference, however, is so tiny that we can generalize it. However, muscle strength varies among individuals based on a variety of elements, including their physical conditions, motivation, physical ability, nutritional status, and genetic factors  . Thus, it is difficult to measure strength without taking these factors into account. This illustrates a limitation of human research. Another limitation of the present study was that the participants were not hospitalized patients or institutionalized individuals. They were volunteers interested in a health study related to BMD measurement. Thus, the present subjects could have been biased towards healthy people or health-conscious individuals. Nevertheless, a novel finding suggesting that the continuous consumption of Ca-supplemented lemon beverages is effective in preventing the loss of BMD after menopause was obtained. This conclusion was based on the changes that occurred after five months of intervention in both bone metabolism markers and the BMD of the lumbar spine and femur. We expect our new discovery to become instrumental in developing preventive strategies for osteoporosis in the future. Research conducted by Nii et al.  has previously demonstrated that, in rats, the consumption of shirasuboshi together with sudachi (Citrus sudachi) juice leads to significant decreases in the concentrations of urinary pyridinoline and deoxypyridinoline and inhibits bone resorption. They have also reported that, in healthy male students, a diet containing shirasuboshi and sudachi juice promotes the absorption of minerals such as Ca and magnesium  . These observations suggest that citric acid in lemons also increased the absorption of dietary Ca in the present study. Further dietary surveys are necessary to substantiate this hypothesis.
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.
Conflicts of Interest
The authors declare no conflicts of interest regarding the publication of this paper.
Cite 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