Young adults achieve peak bone mass around the age of 30 years. Adequate nutrition is thought to be critical in attaining this. Young Indian women may suffer malnutrition and therefore attain an inadequate peak bone mass. For example, they have a low dietary intake of calcium and poor vitamin D status [1
]. The consequence of these deficiencies is likely to be low bone mineral density [3
], and this may predispose to fracture risk in later life. It is estimated that in India, 50 million people have either osteoporosis of low bone mass [3
] and among young Indian women 44% have osteopenia [3
Bone turnover markers (BTM) may be used to assess bone health, and they are particularly useful in clinical trials [4
]; in the presence of low dietary calcium and vitamin D, these markers are usually elevated. High levels of BTM are associated with higher fracture risk in the older woman [5
]. There is evidence that these markers may be higher in Indians than in Western populations. The bone turnover markers recommended as reference markers by the International Osteoporosis Foundation are PINP and CTX [6
Clinical trials of calcium and vitamin D supplementation in young women from a number of countries have shown that there is a consequent decrease in bone turnover markers [7
]. Similar trials on older women have shown benefits for fracture risk [10
Micronutrients, including selenium, zinc, vitamin B12, vitamin K, and folic acid, may play a role in the bone health of young women. Thus, a nutrient supplement that contained all these components might result in an improvement in bone health. Vitamin K increases the ratio of carboxylated to undercarboxylated osteocalcin, and it also increased the amount of protein specific to bone [11
The aims of this study were to evaluate the nutritional status of young Indian women by their dietary record and biochemical assessments and to examine the effect of a nutritional supplement in a randomised controlled trial. The key conclusions were that young Indian women are deficient in certain nutrients. A nutrient supplement improves bone turnover and micronutrient status in some but not all nutrients tested in young Indian women.
2. Materials and Methods
This was a double-blind, randomised, single-centre, parallel group, controlled study, conducted between May 2017 and January 2018 at B. J. Medical College and Sassoon Hospital, Pune, India (ClinicalTrials.gov
identifier: NCT03155269; study number: 207192). Eligible subjects (25 to 45-year-old premenopausal Indian women) were randomly (1:1) allocated to either the test group (protein-rich beverage powder fortified with multi-micronutrients (MMN)) or the control group (low-protein non-fortified isocaloric beverage powder). The composition of the two products can be seen in Table 1
At the screening visit (Visit 1), subjects provided their written informed consent to participate in the study. Demographics, medical history, current/concomitant medications, general physical examinations, and vital signs were recorded. Height, weight, and body mass index (BMI) were assessed using anthropometric measurements. Urine pregnancy testing was carried out on all female subjects who were of childbearing potential. After that, the subject’s eligibility was determined based on inclusion/exclusion criteria. Eligible subjects were given a diary and weighing scale to record 7-day food intake.
Eligible subjects attended the clinical study site for the baseline visit (Visit 2: 7 to 10 days after the screening visit) and were asked to bring the completed 7-day diary for assessment of dietary intake of calcium, protein, and other key micronutrients. At the baseline visit, subjects were randomly allocated to receive either the test or the reference product at a 1:1 ratio, which was stratified by age (Stratum 1: Age ≥25 years to <35 years; Stratum 2: Age ≥35 years to ≤45 years). The study centre aimed to recruit a target ratio of 50% of subjects in the 25 to 35 years age band and 50% in the 35 to 45 years age band with a minimum of 40% in either age band. The study product was dispensed per the randomisation schedule, with the first dose being taken (orally) under the observation of site staff.
The randomisation schedule was generated by the Biostatistics Department, GSK CH, prior to the start of the study, using validated internal software (SAS, Marlow, UK). Subjects were instructed to take the study product (30 g of powder made up in 200 mL water), twice daily (morning and preferably in the evening) for a total of six months. Subjects attended on-site visits at baseline and at four follow-up visits; visit 3: 1.5 months (45 ± 7 days from baseline), visit 4: 3 months (90 ± 7 days), visit 5: 4.5 months (135 ± 7 days), and visit 6: 6 months (end of treatment: 180 days ± 7 days). Subjects were fasting at baseline, month 3, and month 6 visits, and blood (12-h fasting) and spot urine samples were collected at baseline, 3 months, and 6 months.
The study product was consumed under the observation of site staff during each visit except for visit 1 and visit 6. The last dose had to be consumed the evening before the month 6 visit. In between study visits, the subjects consumed the study product at home and completed a product compliance diary. Random home visits were made by site staff to ensure compliance of product consumption at home. At each visit, subjects were asked to return sachets of study product (empty and unconsumed). Subjects were considered as non-compliant if they consumed less than 80% of the total amount of the recommended dose (for the entire study duration).
The study protocol was approved by the Institutional Ethics Committee. The study was conducted in accordance with the ethical principles that are outlined in the Declaration of Helsinki, the International Council for Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) and all applicable local Good Clinical Practice (GCP) and regulations. Written informed consent was obtained from each participant prior to the performance of any study-specific procedures.
2.2. Study Participants
Women aged 25 to 45 years (inclusive) having good general and mental health with no clinically significant and relevant abnormalities in medical history or upon physical examination, and BMI between 18 and 30 kg/m2 (inclusive), were included in the study. Women who had attained physiological menopause defined as those without a menstrual period for 12 consecutive months, who were pregnant or were intending to become pregnant over the duration of the study, and women who were breastfeeding were excluded from the study. Women with current or regular use of any prescription, over-the-counter vitamin supplements, or herbal medicine, treatment with bisphosphonates (any dose within the previous 2 years) or other medications known to affect bone (within the previous 6 months); history of metabolic bone disease; any hormonal disorders or disturbances; and bone fracture in last 12 months and those who were having more than two units of alcohol per day and/or were smoking were also excluded. Additionally, women who took any other health food drinks/beverages or supplements or had been on supplements within a month prior to study start, and women who used any medication that was known to influence bone mass were excluded from the study. The use of calcium, vitamin D, and multivitamin supplements on a regular basis were to be stopped 2 months before the onset of the trial.
The co-primary outcome variables were the changes from baseline after 6 months of treatment in the bone resorption marker serum C-terminal cross-linking telopeptide of type I collagen (s-CTX-I) and the bone formation marker expressed as ratio of carboxylated osteocalcin to under carboxylated-osteocalcin (c-OC/uc-OC).
The secondary outcome variables were change from baseline after 3 and 6 months of treatment in bone formation markers: serum N-terminal propeptide of type I procollagen (s-PINP), bone alkaline phosphatase (BSAP), serum alkaline phosphatase (s-ALP) and c-OC/uc-OC (at 3 months); change from baseline after 3 and 6 months of treatment in bone resorption markers urinary CTX-1, serum N-terminal telopeptide of type 1 collagen (s-NTX-1), and serum CTX-1 (at 3 months); change from baseline after 3 and 6 months of treatment in calcium status determined by serum calcium, parathyroid hormone (s-PTH), and urinary calcium to creatinine ratio (Ca/CR). Finally, changes from baseline after 3 and 6 months of treatment in other bone metabolism parameters and micronutrients including phosphorus, selenium, vitamin D, folate, vitamin B12, vitamin B6, and zinc were assessed.
Safety assessments included assessment of adverse events (AEs) and serious AEs (SAEs). Other safety assessments included laboratory tests, vital signs, physical examination, and haemoglobin examination.
2.4. Laboratory Methods
The following laboratory tests were performed by enzyme-linked immunosorbent assays: c-OC, ucOC (Takara Bio Inc, Shiga, Japan) and serum CTX (Immunodiagnostic Systems, Boldon, UK), BSAP, serum folate, and vitamin B12. Calcium was measured by spectrophotometry. Electrochemiluminescence methods were used for the measurement of osteocalcin, chemiluminescence for PTH, enzyme-linked immunosorbent assay (ELISA), for NTX and radioimmunoassay for intact PINP. Vitamin B6 and 25-D3 were measured by liquid chromatography with tandem mass spectrometry (LC-MS-MS). Selenium and zinc were measured by inductively-coupled plasma/mass spectrometry (ICP-MS). The tests were performed after a 12-h fast, and the urine sample was a morning spot urine. The volume of blood on each visit was 57.5 mL, and the samples were stored at −70 degrees Celsius until measurement.
2.5. Statistical Analysis
A sample size of 44 subjects per group was needed to provide 90% power to detect a difference of −110 ng/L in s-CTX-1, assuming an SD of 144 based on α report by Kruger et al. [7
] and a two-tailed significance level of α = 0.025. To allow for a 20% dropout rate, 54 subjects (total 108) were randomised per treatment arm.
Similarly, 30 subjects per group were needed to provide 90% power to detect a 12.5 difference (test minus control group) in c-OC/uc-OC, assuming a standard deviation (SD) of 13 based on the study by Binkley et al. [14
] and a two-tailed significance level of α = 0.025. To allow for a 20% dropout rate, a total of 36 participants (total = 72) were to be randomised per treatment arm. However, as the sample size for the first primary endpoint was larger, the overall sample size required for this study was 108 in total.
There were three analysis populations. Randomised subjects were all those subjects who were randomised and might or might not have received the study product. The safety population included all subjects who received at least one dose of the study product. Assessment of efficacy was based on the intent-to-treat (ITT) population, which included all subjects in the safety population who had at least one post product co-primary efficacy assessment (either s-CTX-1 or c-OC/uc-OC).
The co-primary efficacy variables of the study were the change from baseline in bone resorption marker (s-CTX-1) after 6 months and the change from baseline in c-OC/uc-OC after 6 months. The efficacy variables were analysed using analyses of covariance (ANCOVA) including the product group and age strata as fixed effects, and the corresponding baseline value as the covariate. Adjusted means, within-product p-values for each product group, product group difference expressed in percentage change from baseline, and the between-product p-values based on the statistical model described above were calculated. Statistical tests to compare treatments were two-sided and were employed at a level of significance of α = 0.025 for the co-primary endpoints. Assumptions of normality and homogeneity of variances in the ANCOVA model were evaluated after study unblinding. If violations were observed, then either suitable data transformations were performed as a post-hoc sensitivity analysis or a non-parametric Van Elteren test was used, and results were to be compared with the primary analysis results. To visually inspect the treatment effect of s-CTX-1 and c-OC/uc-OC, plots across time (baseline, month 3, and month 6) were displayed with least square (LS) means and ±SE bars, which were obtained from the ANCOVA analysis.
As a post-hoc sensitivity analysis, s-CTX-1 was log transformed (natural logarithm), and the log-transformed data were analysed using the ANCOVA model with the product group and age strata as fixed effects, and the corresponding log transformed baseline value as a covariate. The interpretation of the data was based on the geometric mean ratio, 95% confidence interval, and p-value.
A post-hoc subgroup ANCOVA analysis was performed on one of the co-primary variables, s-CTX-1 by age strata using the ITT population. The ANCOVA model had the product group, strata, and the product and strata interaction as fixed effects, and the baseline value of s-CTX-1 as the covariate. Statistical tests to compare treatments were two-sided and employed a level of significance of α = 0.05.
For the secondary efficacy analyses, a similar protocol was followed, using ANCOVA as described above. The level of significance used was α = 0.05. A post-hoc sensitivity analysis was performed using the following variables: urinary Ca/CR, vitamin D3 (using 25 OH D3), and plasma vitamin B6. These variables were log transformed (natural logarithm) and the log-transformed data were analysed using the ANCOVA model with product group and age strata as fixed effects, and the corresponding log transformed baseline value as a covariate. The interpretation of the data was based on the geometric mean ratio and p-value. The efficacy variable of serum folate was analysed using the non-parametric Van Elteren test stratified by age, and the interpretation of data was based on p-values obtained from this test. All analyses were performed in SAS 9.4 (SAS Institute Inc., Cary, NC, USA).