A longitudinal, quasi-experimental, blinded, and randomized 2-year temporary study was carried out. The study included 190 women aged 35–55 years with LBM from a total population of 817 women from central Mexico. However, during the 2-year experiment, nine women (8) from EG1 and one from EG2 did not conclude the experiment due to the following causes: medical treatment for osteoporosis (2), some of them had no attachment to the treatment (3), incomplete studies like Ca/Cr or densitometry (4). Thus, 181 women finished the study. Informed consent was obtained from all patients. The inclusion criteria were: participants who were not pregnant or nursing during the time of study; none of the subjects were undergoing hormone replacement, using steroids, taking medication for bone metabolism, kidney or liver disease or taking vitamin supplements; participants were either premenopausal or menopausal for less than 10 years (either naturally or through surgical procedure); and finally women undergoing osteoporosis treatment or hormone therapy, taking supplements or no longer willing to participate in the study were eliminated. According to the aforementioned criteria, four groups were formed: normal group (NG) that included women with normal BMD as reference population by DXA (n=50), a control group (CG), including women with normocalciuria (n=66) and finally the two experimental groups with hypercalciuria (n=65), called experimental group 1 (EG1, n=29) and experimental group 2 (EG2, n=36) that were supplemented with DN. EG1 and EG2 were randomized using a C++ program.

Nopal pads of O. ficus indica var. redonda were used in this study; the vacuum drying process has been reported elsewhere (12–14) and the physicochemical characterization of DN 600 g is reported in Table 1. It is clear that nopal used in this experiment contained an important amount of calcium without oxalates.

For this treatment, 181 participants were accepted in the trial. They all filled out a medical and nutrition questionnaire, and were provided with next-appointment reminder cards. Taking into account the 24-h recall, diet substitutes were given according to their energy requirements, including all the characteristics of a dietary regimen for osteoporosis.

The recommendations for the consumption of DN are added to the food or preferably mixed with water or natural juices. It is very important to recall that the mean daily value of calcium consumption (24-h recall) of the subject participants in this study was 648±124 mg/day, and that the calcium content of DN 600 g was 3.4 g/100 g. It was necessary to include the recommended 1300 mg/day (mainly from the dairy calcium) and a minimum dose of 2.5 g (67 mg Ca) of DN for CG and EG1, and 800 mg of calcium in the diet with 15 g to reach the recommended 500-mg dose as supplementation in EG2, in order to obtain 1300 mg/day (4). In the case of phosphorous, 1300 mg/day was recommended in order to obtain a Ca/P ratio equal to 1 (9). The complete diet formulation per group is shown in Table 2. For CG, EG1, and EG2, aerobic exercise was recommended such as walking for 30 min at least three times per week, and sun exposure at least 10–15 min/day. In order to determine the compliance of the treatment, there was a monthly follow-up, where women were questioned about the consumption of DN, diet, exercise, and sun exposure. Data were recorded in individual logbooks.

BMD was evaluated at baseline between January and August 2008, and between January and August 2010 through DXA using a Hologic QDR Explorer series densitometer with a variation coefficient of 1.0% using the Hispanic data as reference (22). BMD was measured on the total hip and lumbar spine regions for the diagnosis of LBM; one or both areas were studied in accordance with the official position of the International Society for Clinical Densitometry (from −1.1 to −2.5 SD below the young reference mean) (23). Women with normal BMD meeting inclusion criteria were considered for the DXA reference population and labeled as NG. They were asked not to eat for 12 h prior to the study. Densitometry was performed at baseline and at an annual and 2-year follow-up.

Urine samples were requested at a 2-h collection in the morning for the calcium and creatinine determination through the CCI (from the second morning urine collection and 2 h later, before 10:00 am). The determination of calcium and creatinine was performed using standard methods by photometry absorbance, using Roche Cobas Integra 400 plus software version 2.3 (Windows XP). Depending on the results, if the level of calciuria was ≥0.17 mg/mg, it was regarded as hypercalciuria (24, 25). The calcium/creatinine ratio was analyzed every 6 months during the 2-year study.

Data were analyzed using SPSS Statistics 20.0. Continuous variables were expressed using mean values, whereas categorical variables were expressed using frequency values. Analyses of covariance were employed for testing differences between groups for calciuria and the percentage of difference between BMD₂₀₁₀−BMD ₂₀₀₈ in the total hip, femoral neck, and lumbar spine regions included confounding factors covariates, such as age, high, lean mass, fat mass, and BMD basal. The analysis was done with pre-menopausal and post-menopausal women; a post hoc test was evaluated between group differences. P-values <0.05 were considered statistically significant.

According to the screening process for the 817 women who were studied at the beginning of this experiment, 37.2% were diagnosed with LBM and 6.9% with osteoporosis. The prevalence of both LBM and osteoporosis in women aged 35–49 years was lower than in older women (30.7 and 2.3%, and 45.4 and 18.3%, respectively). Prevalence of both LBM and osteoporosis was higher in the lumbar spine than in the total hip (29 and 7% vs. 15 and 0%, respectively). Meanwhile, the prevalence in the femoral neck region was higher than in the hip (27 and 1%, respectively). The average age at diagnosis was 43 years, 46.6 years for LBM and 51.6 years in women with osteoporosis. The prevalence of hypercalciuria in women with LBM was 37%.

The general characteristics of the 181 women for study groups are given in Table 3.

These data correspond to basal and end characteristics of: age, weight, height, body mass index, lean mass, fat mass, as well as BMD for lumbar spine, total hip, and femoral neck. At the beginning of the study, the NG group shows significantly younger women than other groups. However, weight, height, and BMI did not differ significantly among the groups, and at the end of the trial, these variables follow the same behaviors. For lean mass, at the beginning of the study NG exhibits the highest value, but an important feature in the case of this parameter at the end of the study, this parameter remains constant for all groups during the 2-year experiment. It is well known that this parameter tends to decrease with age. Lean mass is considered as a protective factor for BMD. It means that diet as well as calcium contributed to the maintenance of lean mass in these women (26). Fat mass at the beginning of the experiments did not present significant differences among groups, but at the end of the study, the NG exhibits a significant increase in relation to EG2. This fact can be directly related to the following aspects: the follow-up of the diet, the increase in the fiber consumption (DN), exercise, and the treatment attachment. It is very important to recall that educational counseling about bone health was confirmed in the health of these women. In relation to the prevalence of menopause at the beginning of this study, NG showed 32% of prevalence, while this ranged from 41.4 to 48.6% for groups CG, EG1, and EG2. At the end of the study, the NG showed an increase of 6% (n=3), 2% (n=1) in CG in, 10% (n=3) in EG1, and 2% (n=1) in EG2. It means that only eight women presented menopause during the 2-year follow-up.

BMD shows the baseline BMD of the three studied regions, group by group, which demonstrated a correct diagnosis statistically and clinically. NG presents the BMD within normal ranges and BMD in the control groups and EG1 as well as EG2 show an LBM. It is interesting to observe, that for lumbar spine NG, CG, and EG1 the BMD decreased statistically, while EG2 did not show any statistical difference. In the case of total hip, NG and EG1 did not exhibit statistical differences, while CG and EG2 showed increases in this parameter. For the femoral neck region, all groups exhibited decreases in the BMD.

Figure 1 shows the Ca/Cr index of the different groups per semester. This analysis was done by taking into account menopause stage (pre- and postmenopause), by using analyses of covariance. The covariance was adjusted for the confounding factors: age, BMD basal (lumbar spine, total hip, and femoral neck), height, lean mass, and fat mass, with a post hoc test. Dashed lines in this figure indicate the hypercalciuria limit. It was possible to observe that at the first semester, that calciuria decreased in all study groups, but there were statistical differences in EG1 (post) without hypercalciuria; during the second semester, against EG1 (post) that exhibited hipercalciuria. From a clinic point of view, EG1 (pre) also exhibited hypercalciuria. At the third semester, it is important to observe that all groups remained normal (pre and post), with no statistical difference among them. In the fourth semester, EG1 (post), EG2 (pre and post) showed statistical differences in relation to CG (pre and post) and EG1 (pre). However, EG2 (pre) showed hypercalciuria.

Figure 2 shows the behaviors of lumbar spine, femoral neck, and total hip as a function of the BMD₂₀₁₀−BMD₂₀₀₈ (%) taking into account the menopausal stage (pre and post) using covariance analysis adjusted by age, basal BMD (lumbar spine, total hip and femoral neck), height, lean mass, and fat mass. In the case of lumbar spine there was a decrease in the BMD for all groups and only EG2 (pre) increased statistically. For femoral neck, all groups exhibited a decrease and only CG (pre) increased statistically. In the case of total hip, EG1 and NG showed a decrease in this parameter, and CG and EG2 (post and pre) showed an increase in the BMD.