· Most participants had normal total body bone mineral density-based on Asian reference values.

· Despite having higher body weight, participants had lower total body bone mineral density values than peers from India, China, and Thailand.

· Sex-specific differences were observed in bone turnover markers and regional muscle-bone unit values.

During rapid growth, optimal calcium intake is important to achieve peak bone mass (PBM). Failure to achieve PBM leads to an increased risk of osteoporosis, a major public health issue with huge socioeconomic implications [1,2]. There is an urgent call by the Asian Federation of Osteoporosis Society to implement strategies to tackle the burden of osteoporosis, as Asia is projected to have the highest incidence of osteoporosis worldwide by the year 2050 [3,4]. Thus, it is crucial to initiate preventive measures in early life. The evaluation of bone indices like bone mineral density (BMD), bone mineral content (BMC), muscle-bone unit (MBU) and bone turnover markers (BTMs) is essential for assessing children’s musculoskeletal health to optimize PBM.

The concept of the MBU is critical in understanding the development of children's musculoskeletal health. This concept emphasizes the interdependent relationship between lean body mass (LBM) and bone development [5]. As children grow, their bones must not only lengthen but also adapt to the increasing mechanical loads placed on them by muscle activity. This relationship ensures that bones develop the strength needed to support increased muscle mass and physical activity. Additionally, during bone formation and resorption, the BTMs released into the circulation provide information about the dynamic process of bone metabolism. However, to conduct an accurate assessment and comparison of skeletal health in children, it is important to have access to local data.

Our team conducted a 1-year randomized, double-blind placebo-controlled trial (the PREBONE-Kids study) on the effect of soluble corn fiber and calcium supplementation on bone indices of preadolescent Malaysian children. This trial has led to the publication of several important findings. First, our study demonstrated that calcium supplementation increased the total BMD for the first 6 months [6]. Additionally, we found that LBM is the major determinant of BMD and BMC alongside other modifiable lifestyle factors such as physical activity and calcium intake [7]. Building on the findings and leveraging the rich dataset we compiled, we decided to conduct a secondary analysis of the existing data to establish local reference data for accurate skeletal health assessments that account for unique sex-specific, nutritional and environmental factors. In Malaysia, there are no known data on BMD, BMC, MBU and BTMs among healthy Malaysian children. The International Osteoporosis Foundation has identified the absence of normative BMD data as a critical gap in osteoporosis-prevention efforts [8]. Thus, this study aims to address this gap by conducting a secondary analysis of existing data on bone indices among Malaysian preadolescents aged 9–11 years old.

The study included 127 boys and 116 girls with a mean age of 10.1±1.0 years. The majority of the participants were in Tanner stage I, although 10% of the girls and 1.6% of the boys were in Tanner stage II. The data on weight, height, body composition, dietary intake and serum vitamin D levels have been published previously [9,17,18]. The weight and height of the boys and girls were similar; however, the boys had slightly more LBM. Overall, according to the World Health Organization criteria, 8.6% of the participants were thin for their age, while 17.7% were obese [19]. These study participants habitually consumed 1,457±450 kcal, with 48.9%±6.3% of energy coming from carbohydrates, 17.0%±4.2% of energy coming from protein and 34.6%±6.5% of energy coming from fat (Table 1). Calcium intakes were <400 mg /day, and only 57.6% of the total participants had sufficient vitamin D levels, with a cutoff above 50 nmol/L [20].

The sex-specific and region-specific BMD and BMC values are presented in Table 1. Based upon the Asian reference population [12], 97.5% of the participants exhibited TBBMD z-scores higher than -1 SD, indicating normal bone density. A small percentage (2.5%) of the study participants was identified as being at risk for low TBBMD for their age, falling within the range of -1.9 to -1.0 SDs. However, with only 6 participants in this group, it was not possible to make statistically significant comparisons. Observationally, those children at risk for low TBBMD for their age were noted to have smaller statures and a leaner physique, with their BMI z-scores falling within the negative range. No other differences were observed in their dietary intake or physical activity levels. Notably, none of the participants had severely low TBBMD for their age of below -2 SDs.

Comparatively, based upon the Western reference population [13], the majority of the participants (85.2%) had normal TBBMD for their age, while 14% were at risk of low bone mass and 0.8% had low bone mass for their age.

Despite having a similar body size and bone area, the boys had significantly higher TBBMD and TBBMC compared to the girls. Region-specific BMD values showed that there were no sex-specific differences in the LSBMD, lumbar spine BMC or LS area, with the exception of that boys had higher head and ribs BMD values than the girls.

A sex-specific comparison of MBU is shown in Table 2. While there was not a significant sex-based distinction in total body MBU, it is worth noting that girls exhibited slightly higher MBU values in areas such as the total body less head, arms, legs and android. The LBM was higher among the boys (22.50±5.40 kg) compared to the girls (21.00±5.07 kg, P=0.026). In terms of differences in BTMs, boys had significantly lower OC, PINP and iPTH levels than girls (Table 3).

We compared our TBBMD data with published data of other Asian children that used similar DXA machines from the same manufacturer (GE Lunar Corp., USA). Table 4 presents the comparison of weight and TBBMD values of Malaysian, Thai, Chinese and Indian children of 10 years of age [21-23]. In general, the participants from this study had lower TBBMD values than children of the same age from India, China and Thailand, despite having higher body weights.

This study is the first study to characterize the bone mass and body composition of healthy preadolescent Malaysian children aged 9–11 years using the DXA. We showed that preadolescent Malaysian children have a normal bone mass for their age based on TBBMD z-scores using the Asian reference population database. A greater percentage of children were classified as having a low bone mass for their age when compared to Caucasian cutoff points instead of Asian cutoff points. As TBBMD z-score is the mean of ethnic-specific and sex-specific normative BMD values, it can differ by as much as 2 SDs depending on the normative data used [24]. The data from this study meet the need to have an age, sex and ethnic-specific reference database for the correct interpretation of bone density in growing Malaysian children.

We demonstrated sex-specific differences in the TBBMD and TBBMC despite finding similar bone areas in boys and girls. The boys in our study had higher TBBMD values than the girls, which can be attributed to the higher LBM observed in boys. LBM was identified as a major contributor to TBBMD in our study population, as previously reported [7]. LBM has the strongest effect on TBBMD, with more than half of the differences in TBBMD being attributed to LBM, fat mass and physical activity in the boys and lean mass and calcium intake in the girls, respectively. [7].

Despite sex-specific differences in the TBBMD and TBBMC, there was no difference in the total body MBU of the children. Conversely, studies showed that girls have higher total body MBU values than boys as girls accrue more BMC due to the effect of hormones during puberty [25-27]. However, these studies enrolled older children (>12 years of age) who had attained puberty. The activation of sexual hormone production is known to induce the growth of bone and muscle mass in puberty [28]. As our children were yet to attain puberty, expected sex-specific differences in total body MBU may not be apparent. However, the observed differences in region-specific MBUs in the study participants could be due to a sexual dimorphism in bone mass that is evident in younger children and is likely attributable to differences in LBM [29].

Comparing our data with other population studies using the same DXA equipment, we found that our children's TBBMD values were lower than the age-matched values from India, China, and Thailand. As per our previous study, these differences may be influenced by variations in body composition, along with other factors such as calcium intake, sun, and physical activity levels [7]. Even though body weight improves bone mineralization by exerting mechanical load on weight-bearing bones, the most important weight component for bone density is LBM compared to fat mass [7,30,31]. While our children had higher body weights compared to those from other countries, we were unable to assess or compare differences in LBM and fat mass across the studied populations due to the lack of available data. Future studies assessing BMD and BMC in children should include comprehensive assessments of body composition and lifestyle factors to better understand their roles in BMD across different populations.

Our previous findings established that calcium intake was a significant predictor of BMD and BMC among the girls in our study [7]. Notably, the calcium intake of both the boys and girls in our study ranged from 218–450 mg/day, which is considerably lower than the intake reported in other countries (400–900 mg/day) [32]. Calcium intakes of <450 mg/day have been associated with a deficit of up to 80 g/year in bone calcium deposition [33]. Although physiological adaptations to low dietary calcium intake can enhance calcium absorption efficiency [34], these adaptations are insufficient to compensate for the effects of such low intake levels. Therefore, it can be postulated that, if our participants had consumed higher levels of calcium, their BMD values might have been comparable to those of children in other Asian countries.

In addition to low calcium intake, approximately half of our study participants were found to have low serum vitamin D levels. This is consistent with previous reports of rates of vitamin D inadequacy between 2.8%–65.3% in Eastern Asia, 5.0%–66.7% in Southern Asia, 4.0%–45.5% in Western Asia and 38.1%–78.7% in Central Asian countries [35]. Most of the girls in this study maintained a conservative clothing style while being outdoors, allowing exposure to the sun only on the face, hands and fingers alone, resulting in a low level of sun exposure and low serum vitamin D levels. However, the mean serum vitamin D level was higher than the reported 30 nmol/L, which is the level often associated with osteomalacia and rickets [36]. Furthermore, there is insufficient evidence to indicate that serum vitamin D levels need to be above 50 nmol/L for optimizing BMD [37]. It has been shown that serum vitamin D levels do not affect calcium use in children at peak pubertal growth [38,39].

The sex-specific differences found in the BTM values correspond with findings from Mora et al. in 1998 showing that girls have a significantly higher level of BTMs compared to boys [40]. The BTMs in boys tend to increase much later, as they are higher during early puberty [41] and boys reach puberty much later than girls. A comparison of BTM levels with children of similar ages in other countries was not possible due to a lack of available data. Thus, this study gives the preliminary reference values of BTM levels in healthy preadolescent Malaysian children.

A limitation of the present study is its cross-sectional nature, which provided a snapshot of the bone status of the children rather than a longitudinal follow-up on the effects of dietary and lifestyle behaviors on bone accrual with growth. We also included predominantly Malay children due to the recruitment setting in national primary schools, which fewer children of Chinese and Indian ethnicities typically attend. Ethnicity plays a role in bone mass acquisition during childhood due to differences in calcium-retention rates, body composition, the timing of linear growth and skeletal maturation [42]. The inclusion of children with a wider range of age and sexual maturity stages would also be beneficial, as the PBM-accrual rate occurs within the first year after peak velocity in puberty [43]. However, the strength of this study was that all participants were from the same socio-demographic background and had similar dietary and lifestyle practices, which reduced the environmental confounding factors in bone mineralization.

In conclusion, this study provides normative reference data on the bone mass, BTMs and MBU of healthy preadolescent Malaysian children 9–11 years old. This data will be useful for the assessment and identification of children at risk of fracture. It also adds to the much-needed local and Asian children's BMD reference database. The existence of these reference values allows for comparisons between different populations in Asia and is valuable for use in future studies investigating factors that might improve PBM attainment in children.